SUPPLEMENTARY INFORMATION

Size: px
Start display at page:

Download "SUPPLEMENTARY INFORMATION"

Transcription

1 doi:.38/nature899 Supplementary Figure Suzuki et al. a c p7 -/- / WT ratio (+)/(-) p7 -/- / WT ratio Log X 3. Fold change by treatment ( (+)/(-)) Log X b Fold change by treatment ( (+)/(-)) Fold change (ADR (+)/(-) ) p7 -/- / WT <.66 >.66 ratio (p7- dependent) Supplementary Figure. Comparison of DNA damage-induced mirnas and /7-regulated mirnas. a. mirnas were sorted by p7-dependency (p7 -/- versus WT ratio) from the dataset of mirna microarray expression profile of p7 -/- and WT embryos (E-MEXP-5) 7, and compared to the expression profile of DNA damage ()-induced mirnas in TOVG cells containing wild-type 5. b. Comparison of induction ratio of p7-dependent (p7 -/- /WT <.66) and p7-independent mirnas (p7 -/- /WT >.66) by DNA damage. p7-dependent (p7 -/- /WT <.66) and p7-independent mirnas (p7 -/- /WT >.66) were defined by the dataset of mirna microarray expression profile of p7 -/- and WT embryos (E-MEXP-5) 7, and the induction ratio of each group by DNA damage () was compared and displayed in a boxplot. c. A set of putative /7-dependent mirnas induced by DNA damage. Data from comparison in Fig. Sa was shown. mirnas indicated by asterisks were focused in this study. Log X

2 doi:.38/nature899 mature 3a mature 7 WT -/ U6 snrna pre-6 (long exposure) pre-6 mature 6 mature 5a WT -/ U6 snrna pre-6 (long exposure) pre-6 mature 6 WT -/ U6 snrna WT -/ pre-3 (long exposure) pre-3 mature 3 U6 snrna pre-5 (long exposure) pre-5 mature 5 mature 6a WT -/ U6 snrna Supplementary Figure. Northern blot analysis of mirnas. Total RNA from WT and -/- HCT6 cells with/without treatment were purified, and each RNA was detected by specific probes as mirnas or U6 snrna. Northern blot analysis confirmed the upregulation of pre- and mature mirna levels in response to treatment.

3 doi:.38/nature899 Supplementary Figure 3 a WI-38 cells Mature mirna 3a 5a 6 5b 3a 6a Suzuki et al. hr 8hr hr Precursor mirna 3a 5a 6-5b 6-3a 6a hr 8hr hr Primary mirna 3a 5a 6-5b 6-3a 6a- 6a hr 8hr hr b TIG-3 cells Mature mirna 3a 5a 6 5b 3a 6a hr 8hr hr Precursor mirna 3a 5a 6-5b 6-3a 6a hr 8hr hr Primary mirna 3a 5a 6-5b 6-3a 6a- 6a Supplementary Figure 3. Enhanced maturation of mirnas by DNA damage in human diploid cell lines (WI-38 and TIG-3). a, b. The expression levels of primary precursor, precursor and mature forms of indicated mirnas were examined in two human diploid cell lines, WI-38 cells (a) and TIG-3 cells (b), after treatment with (8 h or h). Error bars represent s.e.m. We observed an upregulation of precursor and mature forms of 5a, -6, -6a, -3, -3, -5, -3 and -6 in WI-38 and TIG-3 cells, in response to treatment (3a is a known transcriptional target). hr 8hr hr 3

4 doi:.38/nature899 Supplementary Figure Suzuki et al. TOVG cells Mature mirna 3a 5a 6 5b 3a 6a hr 8hr hr Precursor mirna 3a 5a 6-5b 6-3a 6a hr 8hr hr Primary mirna 3a 5a 6-5b 6-3a 6a- 6a hr 8hr hr Supplementary Figure. Enhanced maturation of mirnas by DNA damage in WT TOVG cells. The expression levels of primary precursor, precursor and mature forms of indicated mirnas were examined in TOVG cells after treatment with (8 h or h) (P <.5, compared to none; n = 3). Error bars represent s.e.m. Compared to the results in HCT6 cells, post-transcriptional maturation of 3a and -6a was additionally observed in this cell line.

5 doi:.38/nature899 Supplementary Figure 5 a pri-5a pre-5a Mature 5a Suzuki et al. (-) Control p7 Control p7 Control p7 b pri pre Mature 6- (-) Control p7 Control p7 Control p7 c pri-3 pre-3 Mature (-) Control p7 Control p7 Control p7 d pri-6 pre-6 Mature (-) Control p7 Control p7 Control p7 Supplementary Figure 5. Post-transcriptional regulation of mirna maturation by and /p7 under DNA damage. a, b, c, d. WT HCT6 cells were transfected with control or for, or p7. After treatment, the expression levels of primary, precursor and mature mirnas were compared (P <.5, compared to control ( (-)); n = 3). Data for 5a (a), -6- (b), -3 (c) and -6 (d) are shown. 5

6 doi:.38/nature899 Supplementary Figure 6 Suzuki et al. a Relative MTT value day day 3 day 5 NTC a 6 Percetage of cells S G/M NTC a 6 b IB: CDK6 α-tubulin IB: K-Ras α-tubulin NTC 6 NTC 3 6a Relative expression a NTC Relative expression Relative expression NTC 6a NTC 7 Relative expression Relative expression Relative expression NTC 3 NTC 5 NTC c Supplementary Figure 6. Effects of mirnas induced by DNA damage on cell growth and cell cycle. a. Effects of mirnas induced by DNA damage on cell growth and cell cycle of HCT6 cells. Representative data are shown from triplicate experiments. NTC: negative control. b. Immunoblot analysis for validation of mirna targets, K-Ras and CDK6. Putative target sequences in the 3 UTR and validation of its responsiveness are demonstrated in Supplementary Fig. 7 and 8, respectively. NTC: negative control. c. In the experiments in panel (a) and (b), cells were transfected with pre-mir mirna precursors ( nm) (Ambion). Overexpressed mirna levels were confirmed by qrt-pcr analysis. The expression levels of overexpressed mirnas were about 5--fold higher than those of endogenous mirnas. 6

7 doi:.38/nature899 a K-Ras 3'UTR k k 3k k 3 3 5'...UCAUGUUAAAAGAAG-UCAUCUCA... 3' ACUCGAUGUCACGAAGUAGAGU 5'...ACAGUUUGCACAAGU--UCAUCUCA... 3' ACUCGAUGUCACGAAGUAGAGU b CDK6 3'UTR k 3/7 k 5 (A) 3k 6 (A) 5'...CACACAUACCCUCUCUGCUGCUU... 3' GCGGUUAUAAAUGC-ACGACGAU 6 (B) 5'...GAGGUCAGAGUGCCC-UGCUGCUG... 3' GCGGUUAUAAAUGCACGACGAU 6a (A) 5'...UACCCGUUGUACCCU----UACUUGAA... 3' CGGAUAGGACCUAAUGAACUU 6a (B) 5'...UACUGUAUUUCUUCAUACUUGAA... 3' CGGAUAGGACCUAAUGAACUU 3/7 5'...UUUUUCUAACCUUGA--AUGCUGCC... 3' ACUAUCGGGACAUGUUACGACGA 5 (A) 5'...AAUGCAGCUGUUCUGAACUGGAA... 3' UUCCCUAAGGACCCUU-UUGACCUG 5 (B) 5'...CAAUAAUCCUUUGGAAACUGGAU... 3' UUCCCUAAGGACCCUUUUGACCUG 6 5'...UCAAGAAUAUAAACCACAUUCCA... 3' CCGUUUCUUCCU-----UGUAAGGA k 5k 6 (A) 6k 6 (B) 7k 6a (A) 8k 6 Supplementary Figure 7. Identification of target genes regulated by DNA damage-induced mirnas. 9k 6a (B) 5 (B) a, b. Bioinformatic analysis identified K-Ras (a) as a novel potential target of 3 and CDK6 (b) as common targets of 6/-6a/-7/-5/-6. The putative mirna target sequences in K-Ras and CDK6 3 UTRs, which are complementary to the mirnas seed region, are shown. 7

8 doi:.38/nature899 Supplementary Figure 8 a K-Ras 3 UTR k k 3k k b 3 A B C CDK6 3 UTR 3 (P) 3 : conserved site (P): poorly conserved site Luciferase activity (relative level) a control KRas-A KRas-B KRas-C : conserved site (P): poorly conserved site NTC 3 k k 3k k 5k 6k 7k 8k 9k 6 3/7 5 (P) 6 (P) 6 (P) 6a 6a A 5 3/7 C D (P) E B. control. Cdk6-A. Cdk6-B Luciferase activity (relative level) Luciferase activity (relative level) Luciferase activity (relative level) NTC 6 6a NTC 6 6a NTC 6 6a Cdk6-C. Cdk6-D. Cdk6-E Luciferase activity (relative level) Luciferase activity (relative level) Luciferase activity (relative level) NTC 6 6a NTC 6 6a NTC 6 6a Supplementary Figure 8. Validation of K-Ras and CDK6 as targets of 6/-6a/-7/-3/-5/-6. a, b. 3 UTR fragments of K-Ras (a) and CDK6 (b) containing potential mirna target sites were cloned into 3 UTR of the luciferase gene in the Psicheck dual luciferase reporter vector (Promega). HCT6 cells were then transfected with each reporter construct with/without pre-mir precursors and analyzed for the measurement of luciferase expression (P <.5, compared to negative control (NTC); n = 3). Error bars represent s.e.m. Overexpression of 6, -6a, -3, -3, -5 and -6 decreased reporter expression containing the corresponding 3 UTR target sites, indicating that these potential 3 UTR target sites are functional as target of mirna-mediated gene regulation. 8

9 doi:.38/nature899 Supplementary Figure 9 a. K-Ras 3 UTR k k 3k k 3 3 (P) 3 Luciferase activity (relative level).8.6. (-) (+) A B. C : conserved site (P): poorly conserved site control KRas-A KRas-B KRas-C b CDK6 3 UTR 6a : conserved site (P): poorly conserved site k k 3k k 5k 6k 7k 8k 9k 6 3/7 5 (P) 6 (P) 6 (P) 6a 6a A 5 3/7 C D (P) E B. Luciferase activity (relative level) (-) (+) control CDK6-A CDK6-B CDK6-C CDK6-D CDK6-E Supplementary Figure 9. Attenuation of K-Ras and CDK6 3 UTR activities under DNA damaging condition. a, b. HCT6 cells were transfected with each reporter construct (a; K-Ras, b; CDK6). After treatment, the luciferase expression was examined (P <.5, compared to (-); n = 3). Error bars represent s.e.m. The expression from UTR reporter was significantly decreased by treatment, while that of control reporter was not affected by DNA damage. 9

10 doi:.38/nature899 a IB: MCF7 cells input (/) Control IgG DROSHA Ab DROSHA a-tubulin IB: b DROSHA Control IgG DROSHA Ab input (/) DGCR8 α-tubulin WI-38 cells IB: c DROSHA Control IgG DROSHA Ab input (/) DGCR8 α-tubulin TIG-3 cells Supplementary Figure. interacts with DROSHA in various cell lines upon treatment. a, b, c. Immunoprecipitation assays were performed to detect interaction between endogenous and DROSHA in -competent MCF7 (a), WI-38 (b) and TIG-3 (c) cells treated by (8 h). IP, immunoprecipitation; IB, immunoblot.

11 vector ΔTAD ΔPRD ΔDBD ΔDBD-N ΔDBD-C ΔC doi:.38/nature899 a ΔTAD ΔPRD ΔDBD ΔDBD-N ΔDBD-C ΔC TAD PRD DBD C b 6Myc- FLAG-DROSHA input (/) Myc FLAG IP: FLAG Myc FLAG Supplementary Figure. interacts with DROSHA through the DNA-binding domain. a, b. Regions in responsible for the association with DROSHA were determined. Flag-tagged DROSHA was co-transfected into HCT6 cells with the indicated Myc-tagged constructs and immunoprecipitated with anti-flag antibody. Schematic diagram of wild-type (shown on top) and the deletion constructs is shown in the upper panel (a). The transactivation domain, proline-rich domain, DNA-binding domain and the C-terminus are indicated as TAD, PRD, DBD and C, respectively.

12 vector ΔTAD ΔPRD ΔDBD ΔDBD-N ΔDBD-C ΔC doi:.38/nature899 6Myc- FLAG input (/) Myc FLAG IP: FLAG Myc FLAG Supplementary Figure. interacts with RNA helicase through the DNA-binding domain. Regions in responsible for the association with RNA helicase were determined. Flag-tagged was co-transfected into HCT6 cells with the indicated Myc-tagged constructs and immunoprecipitated with anti-flag antibody. Schematic diagram of wild-type and the deletion constructs is shown in Supplementary Fig. a. In consistent with -dependency of DROSHA- association (Fig. d), the interaction between and is also mediated through a carboxyl-terminal half of central DNA-binding domain, as well as DROSHA- interaction (Supplementary Fig. ).

13 doi:.38/nature899 Supplementary Figure 3 Suzuki et al. a WT HCT6 cells unprocessed pri-mirna hr 8hr hr 3a 5a 6-5b 6-3a 6a- 6a b TOVG cells unprocessed pri-mirna hr 8hr hr 3a 5a 6-5b 6-3a 6a- 6a Supplementary Figure 3. Reduction of unprocessed pri-mirna levels as the expense of upregulation of pre- and mature mirnas. a, b. The primer sets were designed for evaluation of unprocessed pri-mirnas, in which one primer covers the pre-mirna and the other covers its franking region. The expression levels of unprocessed pri-mirnas were examined in HCT6 cells (a) and TOVG cells (b) after treatment with (8 h or h). The expression levels of the unprocessed pri-mirnas (5a, -6-, -3-, -3, -5 and -3) decreased in contrast to the upregulation of pre- and mature mirnas, in response to DNA damage. On the other hand, the unprocessed pri-mir of 3a increased, reflecting its transcriptional activation by. The reduced unprocessed pri-mir level and increased expression of pre- and mature mirna suggests that DNA damaging conditions resulted in enhanced DROSHA-mediated pri-mir cleavage. 3

14 doi:.38/nature899 a HCT6 cell RNA-ChIP binding (relative to IgG control) 8 6 RNA-ChIP binding (relative to IgG control) 5 5 (-) IP DROSHA IgG -RT IP DROSHA IgG -RT b HCT6 cell 6-3 RNA-ChIP binding (relative to IgG control) 8 6 RNA-ChIP binding (relative to IgG control) Control Control + + IP DROSHA IgG -RT IP DROSHA IgG -RT Supplementary Figure. Associations between several pri-mirs and DROSHA/ are enhanced in a -depedent manner in response to DNA damage in HCT6 cells. a, b. RNA-ChIP analysis for association between pri-6-/-3 and, or DROSHA in HCT6 cells. After treatment of HCT6 cells with (8 h), endogenous, or DROSHA were immunoprecipitated and subjected to RT-PCR analysis (a). As controls, RNA samples untreated with reverse transcriptase (-RT) or immunoprecipitated with non-specific IgG (IgG) were subjected to PCR (P <.5 compared to none or control ( (-)); n = 3). After transfection with control or, similar analysis was performed (b).

15 doi:.38/nature899 %of Max 8 6 negative control (no infection) Control DGCR8 3 GFP intensity DROSHA function Supplementary Figure 5. Monitoring of DROSHA function in vivo. Effect of DGCR8 knockdown on GFP intensity. GFP signals were elevated by DGCR8 knockdown. High responder clones to DGCR8 were used for further analysis. 5

16 doi:.38/nature899 Supplementary Figure 6 Suzuki et al. a ( -/- HCT6 cells) vector (-) null WT C35Y R75H R73H b ( -/- HCT6 cells) Control pri-6- pre-6- mature 6 pri-6- pre-6- mature 6 c.5 6 ( WT HCT6 cells).5.5 Control Control pri-6- pre-6- mature 6 Supplementary Figure 6. Effects of the wild-type and mutant on expression of 6 production from ectopically expressed pri-6 construct. a, b. -/- HCT6 cells carrying CMV promoter-driven pri-6- were infected with adenovirus carrying wild type (WT) or tumor-derived transcriptionally inactive mutant (C35Y, R75H and R73H) or control adenovirus (Ad-null) (a), or transfected with control or (b). The amounts of primary, precursor and mature 6s were examined (n = 3). Error bars represent s.e.m. c. WT HCT6 cells carrying CMV promoter-driven pri-6- were transfected with control, or. The amounts of primary, precursor and mature 6s were examined following treatment. Elevation of pre- and mature mirna was observed in a /-dependent manner. 6

17 doi:.38/nature899 a b.6 3 ( -/- HCT6 cells) 3 ( -/- HCT6 cells) vector (-) null WT C35Y R75H R73H Control pri-3 pre-3 mature 3 pri-3 pre-3 mature 3 c d 3 ( WT HCT6 cells) 3 ( WT HCT6 cells) Control Control pre3 mature 3 U6 snrna Control pri-3 pre-3 mature 3 Supplementary Figure 7. Effects of the wild-type and mutant on expression of 3 production from ectopically expressed pri-3 construct. a, b. -/- HCT6 cells carrying pmxs-puro-chimera-3-egfp were infected with adenovirus carrying wild type (WT) or tumor-derived transcriptionally inactive mutant (C35Y, R75H and R73H) or control adenovirus (Ad-null) (a), or transfected with control or (b). The amounts of primary, precursor and mature 3s were examined (n = 3). Error bars represent s.e.m. c, d. WT HCT6 cells carrying pmxs-puro-chimera-3-egfp were transfected with control, or. The amounts of primary, precursor and mature 3s were examined following treatment. Elevation of pre- and mature mirna was observed in a /-dependent manner and confirmed by northern blot analysis (d). 7

18 doi:.38/nature899 WT C35Y 8 8 % of Max 6 % of Max R75H R73H 8 8 % of Max 6 % of Max monitor vector (-) Ad-null GFP intensity Supplementary Figure 8. Effects of the wild-type and mutant on DROSHA function in vivo. FACS analysis of -/- HCT6 cells harboring pmxs-puro-egfp-5/3. Changes of GFP signals were evaluated after Ad- (WT, C35Y, R75H and R73H) infection or transfection with s. Mean fluorescence intensities were summarized in Fig. d. 8

19 doi:.38/nature899 Supplementary Methods RT-PCR primers. The primer sequences used were as follows. : These primers were previously designed in the following report. Jiang, J., Lee, E. J., Gusev, Y. & Schmittgen, T. D. Real-time expression profiling of microrna precursors in human cancer cell lines. Nucleic Acids Res 33, (5). Gene Primer sequence Reference human pre-3a human pre-5a human pre-6- human pre-5b human pre-6- human pre-3a human pre-6a human pre-3 forward 5 -TGGCAGTGTCTTAGCTGGTTG-3 reverse 5 -GGCAGTATACTTGCTGATTGCTT-3 forward 5 -CAGCACATAATGGTTTGTGGA-3 reverse 5 -GCAGCACAATATGGCCTG-3 forward 5 -GCAGCACGTAAATATTGGCGT-3 reverse 5 -CAGCAGCACAGTTAATACTGGAGA-3 forward 5 -AGCACATCATGGTTTACATGC-3 reverse 5 -CTAGAGCAGCAAATAATGATTCG-3 forward 5 -GCACGTAAATATTGGCGTAGT-3 reverse 5 -AAGCAGCACAGTAATATTGGTG-3 forward 5 -CTGGGGTTCCTGGGGAT-3 reverse 5 -TGGAAATCCCTGGCAATGTG-3 forward 5 -TTCAAGTAATCCAGGATAGGCTGT-3 reverse 5 -TGCAAGTAACCAAGAATAGGCC-3 forward 5 -GCTTCTTTACAGTGCTGCCT-3 reverse 5 -TTCATAGCCCTGTACAATGCT-3 9

20 doi:.38/nature899 human pre-7 forward 5 -CAGCTTCTTTACAGTGTTGCCT-3 reverse 5 -GATAGCCCTGTACAATGCTGC-3 human pre-3 human pre-5 human pre-8 human pre-3 human pre-6 human pre- human pri-3a human pri-5a human pri-6- human pri-5b human pri-6- forward 5 -TGAGGTGCAGTGCTGCATC-3 reverse 5 -GCTACAGTGCTTCATCTCAGACTC-3 forward 5 -GTCCAGTTTTCCCAGGAATC-3 reverse 5 -AGAACAGTATTTCCAGGAAT-3 forward 5 -TTTGGCAATGGTAGAACTCAC-3 reverse 5 -GTTGGCAAGTCTAGAACCACC-3 forward 5 -TCCAGTGGTTCTTAACAGTTCA-3 reverse 5 -GGTCTAGTGGTCCTAAACATTTC-3 forward 5 -ACATGCTTCTTTATATCCCCA-3 reverse 5 -AAACCACACACTTCCTTACATTC-3 forward 5 -TCTGCCTGTCTACACTTGCTG-3 reverse 5 -TGACTGCCTGTCTGTGCCT-3 forward 5 -CCTCCAAGCCAGCTCAGTTG-3 reverse 5 -TGACTTTGGTCCAATTCCTGTTG-3 forward 5 -CACACATTCGCGCCTAAAGAA-3 reverse 5 -CCTATAGCACTGTGCTGGGCA-3 forward 5 -GCAATTACAGTATTTTAAGAGATGAT-3 reverse 5 -CATACTCTACAGTTGTGTTTTAATGT-3 forward 5 -CAGAACGGCCTGCAGAGATAAT-3 reverse 5 -TGGTCCAAGTCAATTCCATGG-3 forward 5 -CTGTACACTTGGTGTTTG-3 reverse 5 -GTAGTAGTGGTTCCACCA-3

21 doi:.38/nature899 human pri-3a human pri-6a- human pri-6a- human pri-3- human pri-3- human pri-7 forward 5 -TCTCATATGCAGGAGCCACCA-3 reverse 5 -GCAAGTTGCTGTAGCCTCCTTG-3 forward 5 -GCCCAATGGCATAGCAAGA-3 reverse 5 -GGCCAGTCATGCTTACAGTCAC-3 forward 5 -AGGCATCCCTGTGATGAAGGT-3 reverse 5 -TTGAGAGCTCCTTGCTGCTGT-3 forward 5 -AATGCAAGCTCCACAAAGCC-3 reverse 5 -CCAGATGCCAGACATCGTTCT-3 forward 5 -GAGGAAGAGTGGAAGGTAGCCA-3 reverse 5 -AGCATCGTTATCCATCATCACC-3 forward 5 -GCAAGTCGAGCATTTTACCTGC-3 reverse 5 -GCCATGTGTCCACTGAAATGTG-3 forward (F) 5 -GAAGTTGTTCTGCAGCCATCA-3 human pri-3 reverse (R) 5 -TCTGTCCTTCCTGTTATGGCC-3 forward (F) 5 -CAAGGTTTGGTCCTGGGTGCTCAAA-3 reverse (R) 5 -TGGTGGCCTGTGGCGGGACTCCAA-3 human pri-5 human pri-8 human pri-3 human pri-6 forward 5 -TGGATTTGCCTCCTTCCCA-3 reverse 5 -TTGAACCCTCATCCTGTGAGCC-3 forward 5 -CTGAAACCAAACATCCCCCA-3 reverse 5 -TCACAGGTCCTCAAGTCAGCTG-3 forward 5 -TCTCCACTCACTGAGGCCTTAG-3 reverse 5 -AGGTCCTTCACGAGTTTAGCG-3 forward 5 -CGTGGTGCTGCTTATCTGTGAA-3 reverse 5 -TGTTGTGAGGAATGGCAGTGC-3

22 doi:.38/nature899 human pri- human β-actin forward 5 -TATTTCATAGGCACCACTCACTTT-3 reverse 5 -CATAGCATCTACACCAAGAGCTAC-3 forward 5 -TCACCCACACTGTGCCCATCTACGA-3 reverse 5 -CAGCGGAACCGCTCATTGCCAATGG-3 human U6 snrna forward 5 -CTCGCTTCGGCAGCACA-3 reverse 5 -AACGCTTCACGAATTTGCGT-3 RT-PCR primers for evaluation of unprocessed pri-mirnas. Gene human pri-3a human pri-5a human pri-6- human pri-5b human pri-6- human pri-3a human pri-6a- Primer sequence forward 5 -CAACCAGCTAAGACACTGCCAA-3 reverse 5 -CCTCCTGCATCCTTTCTTTCCT-3 forward 5 -CCATTATGTGCTGCTACTTT-3 reverse 5 -TGCATATTACATCAATGTT-3 forward 5 -CCTCTAATGCTGCATAAGCT-3 reverse 5 -CCAGTATTAACTGTGCTGCT-3 forward 5 -CTATAAAGCTAGGTTGGATG-3 reverse 5 -CTTGACTGTAGCATGTAAAC-3 forward 5 -CGTTTTATGTTTGGATGAACTG-3 reverse 5 -CGCCAATATTTACGTGCTG-3 forward 5 -GGCAATGTGATTTGTGACAGGA-3 reverse 5 -GGAGGTGTCCCCAAATCTCAT-3 forward 5 -AATGAAGCCACAGGAGCCA-3 reverse 5 -TGCACAGCCTATCCTGGATTA-3

23 doi:.38/nature899 human pri-6a- human pri-3- human pri-3- human pri-7 human pri-3 human pri-5 human pri-8 human pri-3 human pri-6 human pri- forward 5 -TACTTGAATCCAGCCACAGCC-3 reverse 5 -TCATGACCTGGACAGACTGTCC-3 forward 5 -AGGCAGCACTGTAAAGAAGCC-3 reverse 5 -AAGGCAGCTATGCTCACCACT-3 forward 5 -AGCATTCAGGTCAAGCAGCATT-3 reverse 5 -AAGCTGCACCAACTGGATCCT-3 forward 5 -TGCTTGAACTCCATGCCACA-3 reverse 5 -GGTCTGAGCATCCTTGCACAT-3 forward 5 -GTGCTGCATCTCTGGTCAGTTG-3 reverse 5 -AGCACTTACCACTTCCAGGCTG-3 forward 5 -GGGATTCCTGGAAATACTGT-3 reverse 5 -CCTCTTACCTCCAGGGACAG-3 forward 5 -ACCGGATCCTGTTACCTCACCA-3 reverse 5 -TCTCTTCCTCAGCACAGACCGA-3 forward 5 -CAGTTCTGTAGCGCAATTGTGA-3 reverse 5 -TGACTGTGACTCTGACTCCAGG-3 forward 5 -AAGGAAGTGTGTGGTTTCGGC-3 reverse 5 -TGGCACAAAGCCCTGATGA-3 forward 5 -ACTTGCTGTGCAGAACATCCG-3 reverse 5 -GCTTTCTTTCAATGGCTGGTTG-3 Northern probes. Small RNA hsa-3a Sequence ACAACCAGCTAAGACACTGCCA 3

24 doi:.38/nature899 hsa-6 hsa-6a hsa-7 hsa-3 hsa-5 hsa-6 U6 snrna CGCCAATATTTACGTGCTGCTA AGCCTATCCTGGATTACTTGAA TGATAGCCCTGTACAATGCTGCT GAGCTACAGTGCTTCATCTCA AGGGATTCCTGGGAAAACTGGAC CCACACACTTCCTTACATTCCA ATATGGAACGCTTCACGAATT RNA-ChIP primers. Human 6- Forward primer: 5'-GCAATTACAGTATTTTAAGAGATGAT-3' Reverse primer: 5'- CATACTCTACAGTTGTGTTTTAATGT-3' Human 3 Forward primer: 5'- CAAGGTTTGGTCCTGGGTGCTCAAA-3' Reverse primer: 5'- TGGTGGCCTGTGGCGGGACTCCAA-3'.

25 doi:.38/nature899 RNAi sequences The target sequences for were as follows: (four kinds of independent duplexes were mixed) #: GAAAUUUGCGUGUGGAGUA #: GUGCAGCUGUGGGUUGAUU #3: GCAGUCAGAUCCUAGCGUC #: GGAGAAUAUUUCACCCUUC (four kinds of independent duplexes were mixed) #: GCAAAUGUCAUGGAUGUUA #: CAACCUACCUUGUCCUUGA #3: GCAUGUCGCUUGAAGUCUA #: CCAAAUAUGCACAAUGGUA p7 (four kinds of independent duplexes were mixed) #: CAAGGAUGGUGGCCGGAGA #: CGAUAGAGCUGGUUAUGCU #3: CAAAUGCAGUGUAGAGCUA #: GGAGUGCAUUUGAUAGUUA 5

26 doi:.38/nature899 Primers for cloning of K-Ras and CDK6 3 UTR. The primer sequences used were as follows. The XhoI-NotI digested PCR products were inserted between the XhoI and NotI sites of the 3 UTR of the luciferase gene in the Psicheck dual luciferase reporter vector (Promega). The underlined nucleotides in the primer sequences indicate XhoI and NotI sites. Gene K-Ras 3 UTR-A K-Ras 3 UTR-B K-Ras 3 UTR-C CDK6 3 UTR-A CDK6 3 UTR-B CDK6 3 UTR-C CDK6 3 UTR-D CDK6 3 UTR-E Primer sequence forward 5 -ccgctcgagaggccctgtgtgaacctttgagcttt-3 reverse 5 -ttgcggccgcacttgaggtcaggagttcgaga-3 forward 5 -ccgctcgagataaacaggtgcctgaactagtt-3 reverse 5 -ttgcggccgcacttcaaactattaaactaaaaca-3 forward 5 -ccgctcgagaggccctgtgtgaacctttgagcttt-3 reverse 5 -ttgcggccgcacttcaaactattaaactaaaaca-3 forward 5 -ccgctcgagggcctcagcagccgccttaagctga-3 reverse 5 -ttgcggccgccaggaaagagtttctgacaaatt-3 forward 5 -ccgctcgagtgcagtccttctacctggtgttt-3 reverse 5 -ttgcggccgcggcaaagatgtttgaaaccatatgt-3 forward 5 -ccgctcgaggatgagctttcttgttttatt-3 reverse 5 -ttgcggccgccatctctttttacccgaacttt-3 forward 5 -ccgctcgagcggtagcagctgccctgtgtgaa-3 reverse 5 -ttgcggccgcctgacagttctgcttctacagaaa-3 forward 5 -ccgctcgagagatgaccaacatccccacacct-3 reverse 5 -ttgcggccgcaagaaatacaaaaggtattacaaa-3 6

ASPP1 Fw GGTTGGGAATCCACGTGTTG ASPP1 Rv GCCATATCTTGGAGCTCTGAGAG

ASPP1 Fw GGTTGGGAATCCACGTGTTG ASPP1 Rv GCCATATCTTGGAGCTCTGAGAG Supplemental Materials and Methods Plasmids: the following plasmids were used in the supplementary data: pwzl-myc- Lats2 (Aylon et al, 2006), pretrosuper-vector and pretrosuper-shp53 (generous gift of

More information

HCT116 SW48 Nutlin: p53

HCT116 SW48 Nutlin: p53 Figure S HCT6 SW8 Nutlin: - + - + p GAPDH Figure S. Nutlin- treatment induces p protein. HCT6 and SW8 cells were left untreated or treated for 8 hr with Nutlin- ( µm) to up-regulate p. Whole cell lysates

More information

Supplementary Table 1. The Q-PCR primer sequence is summarized in the following table.

Supplementary Table 1. The Q-PCR primer sequence is summarized in the following table. Supplementary Table 1. The Q-PCR primer sequence is summarized in the following table. Name Sequence (5-3 ) Application Flag-u ggactacaaggacgacgatgac Shared upstream primer for all the amplifications of

More information

Supporting Online Material for

Supporting Online Material for www.sciencemag.org/cgi/content/full/1137999/dc1 Supporting Online Material for Disrupting the Pairing Between let-7 and Enhances Oncogenic Transformation Christine Mayr, Michael T. Hemann, David P. Bartel*

More information

supplementary information

supplementary information DOI: 1.138/ncb1839 a b Control 1 2 3 Control 1 2 3 Fbw7 Smad3 1 2 3 4 1 2 3 4 c d IGF-1 IGF-1Rβ IGF-1Rβ-P Control / 1 2 3 4 Real-time RT-PCR Relative quantity (IGF-1/ mrna) 2 1 IGF-1 1 2 3 4 Control /

More information

Thermo Scientific Dharmacon SMARTvector 2.0 Lentiviral shrna Particles

Thermo Scientific Dharmacon SMARTvector 2.0 Lentiviral shrna Particles Thermo Scientific Dharmacon SMARTvector 2.0 Lentiviral shrna Particles Long-term gene silencing shrna-specific design algorithm High titer, purified particles Thermo Scientific Dharmacon SMARTvector shrna

More information

Supplementary information to accompany: A novel role for the DNA repair gene Rad51 in Netrin-1 signalling

Supplementary information to accompany: A novel role for the DNA repair gene Rad51 in Netrin-1 signalling Supplementary information to accompany: A novel role for the DNA repair gene Rad51 in Netrin-1 signalling Glendining KA 1, Markie D 2, Gardner RJM 4, Franz EA 3, Robertson SP 4, Jasoni CL 1 Supplementary

More information

Figure 1: TDP-43 is subject to lysine acetylation within the RNA-binding domain a) QBI-293 cells were transfected with TDP-43 in the presence or

Figure 1: TDP-43 is subject to lysine acetylation within the RNA-binding domain a) QBI-293 cells were transfected with TDP-43 in the presence or Figure 1: TDP-43 is subject to lysine acetylation within the RNA-binding domain a) QBI-293 cells were transfected with TDP-43 in the presence or absence of the acetyltransferase CBP and acetylated TDP-43

More information

Supplementary Fig. S1. SAMHD1c has a more potent dntpase activity than. SAMHD1c. Purified recombinant SAMHD1c and SAMHD1c proteins (with

Supplementary Fig. S1. SAMHD1c has a more potent dntpase activity than. SAMHD1c. Purified recombinant SAMHD1c and SAMHD1c proteins (with Supplementary Fig. S1. SAMHD1c has a more potent dntpase activity than SAMHD1c. Purified recombinant SAMHD1c and SAMHD1c proteins (with concentration of 800nM) were incubated with 1mM dgtp for the indicated

More information

Structure, evolution and function of the bi-directionally transcribed iab4 microrna locus in insects

Structure, evolution and function of the bi-directionally transcribed iab4 microrna locus in insects Supplementary data Structure, evolution and function of the bi-directionally transcribed iab4 microrna locus in insects Jerome H.L. Hui, Antonio Marco, Suzanne Hunt, Janet Melling, Sam Griffiths-Jones,

More information

Figure S1. Figure S2. Figure S3 HB Anti-FSP27 (COOH-terminal peptide) Ab. Anti-GST-FSP27(45-127) Ab.

Figure S1. Figure S2. Figure S3 HB Anti-FSP27 (COOH-terminal peptide) Ab. Anti-GST-FSP27(45-127) Ab. / 36B4 mrna ratio Figure S1 * 2. 1.6 1.2.8 *.4 control TNFα BRL49653 Figure S2 Su bw AT p iw Anti- (COOH-terminal peptide) Ab Blot : Anti-GST-(45-127) Ab β-actin Figure S3 HB2 HW AT BA T Figure S4 A TAG

More information

Regulation of Synaptic Structure and Function by FMRP- Associated MicroRNAs mir-125b and mir-132

Regulation of Synaptic Structure and Function by FMRP- Associated MicroRNAs mir-125b and mir-132 Neuron, Volume 65 Regulation of Synaptic Structure and Function by FMRP- Associated MicroRNAs mir-125b and mir-132 Dieter Edbauer, Joel R. Neilson, Kelly A. Foster, Chi-Fong Wang, Daniel P. Seeburg, Matthew

More information

Supplementary Information Design of small molecule-responsive micrornas based on structural requirements for Drosha processing

Supplementary Information Design of small molecule-responsive micrornas based on structural requirements for Drosha processing Supplementary Information Design of small molecule-responsive micrornas based on structural requirements for Drosha processing Chase L. Beisel, Yvonne Y. Chen, Stephanie J. Culler, Kevin G. Hoff, & Christina

More information

GFP CCD2 GFP IP:GFP

GFP CCD2 GFP IP:GFP D1 D2 1 75 95 148 178 492 GFP CCD1 CCD2 CCD2 GFP D1 D2 GFP D1 D2 Beclin 1 IB:GFP IP:GFP Supplementary Figure 1: Mapping domains required for binding to HEK293T cells are transfected with EGFP-tagged mutant

More information

Construction of plant complementation vector and generation of transgenic plants

Construction of plant complementation vector and generation of transgenic plants MATERIAL S AND METHODS Plant materials and growth conditions Arabidopsis ecotype Columbia (Col0) was used for this study. SALK_072009, SALK_076309, and SALK_027645 were obtained from the Arabidopsis Biological

More information

Supporting Information

Supporting Information Supporting Information Su et al. 10.1073/pnas.1211604110 SI Materials and Methods Cell Culture and Plasmids. Tera-1 and Tera-2 cells (ATCC: HTB- 105/106) were maintained in McCoy s 5A medium with 15% FBS

More information

mir-24-mediated down-regulation of H2AX suppresses DNA repair

mir-24-mediated down-regulation of H2AX suppresses DNA repair Supplemental Online Material mir-24-mediated down-regulation of H2AX suppresses DNA repair in terminally differentiated blood cells Ashish Lal 1,4, Yunfeng Pan 2,4, Francisco Navarro 1,4, Derek M. Dykxhoorn

More information

Figure S1. Immunoblotting showing proper expression of tagged R and AVR proteins in N. benthamiana.

Figure S1. Immunoblotting showing proper expression of tagged R and AVR proteins in N. benthamiana. SUPPLEMENTARY FIGURES Figure S1. Immunoblotting showing proper expression of tagged R and AVR proteins in N. benthamiana. YFP:, :CFP, HA: and (A) and HA, CFP and YFPtagged and AVR1-CO39 (B) were expressed

More information

Learning Objectives. Define RNA interference. Define basic terminology. Describe molecular mechanism. Define VSP and relevance

Learning Objectives. Define RNA interference. Define basic terminology. Describe molecular mechanism. Define VSP and relevance Learning Objectives Define RNA interference Define basic terminology Describe molecular mechanism Define VSP and relevance Describe role of RNAi in antigenic variation A Nobel Way to Regulate Gene Expression

More information

Supplementary Figure 1

Supplementary Figure 1 Supplementary Figure 1 Supplementary Figure 1: Vector maps of TRMPV and TRMPVIR variants. Many derivatives of TRMPV have been generated and tested. Unless otherwise noted, experiments in this paper use

More information

Galina Gabriely, Ph.D. BWH/HMS

Galina Gabriely, Ph.D. BWH/HMS Galina Gabriely, Ph.D. BWH/HMS Email: ggabriely@rics.bwh.harvard.edu Outline: microrna overview microrna expression analysis microrna functional analysis microrna (mirna) Characteristics mirnas discovered

More information

Alternative Cleavage and Polyadenylation of RNA

Alternative Cleavage and Polyadenylation of RNA Developmental Cell 18 Supplemental Information The Spen Family Protein FPA Controls Alternative Cleavage and Polyadenylation of RNA Csaba Hornyik, Lionel C. Terzi, and Gordon G. Simpson Figure S1, related

More information

Fatchiyah

Fatchiyah Fatchiyah Email: fatchiya@yahoo.co.id RNAs: mrna trna rrna RNAi DNAs: Protein: genome DNA cdna mikro-makro mono-poly single-multi Analysis: Identification human and animal disease Finger printing Sexing

More information

Supplementary Figure 1. (a) The qrt-pcr for lnc-2, lnc-6 and lnc-7 RNA level in DU145, 22Rv1, wild type HCT116 and HCT116 Dicer ex5 cells transfected

Supplementary Figure 1. (a) The qrt-pcr for lnc-2, lnc-6 and lnc-7 RNA level in DU145, 22Rv1, wild type HCT116 and HCT116 Dicer ex5 cells transfected Supplementary Figure 1. (a) The qrt-pcr for lnc-2, lnc-6 and lnc-7 RNA level in DU145, 22Rv1, wild type HCT116 and HCT116 Dicer ex5 cells transfected with the sirna against lnc-2, lnc-6, lnc-7, and the

More information

Supplementary Figures

Supplementary Figures Supplementary Figures Supplementary Figure 1. Description of the observed lymphatic metastases in two different SIX1-induced MCF7 metastasis models (Nude and NOD/SCID). Supplementary Figure 2. MCF7-SIX1

More information

RNA oligonucleotides and 2 -O-methylated oligonucleotides were synthesized by. 5 AGACACAAACACCAUUGUCACACUCCACAGC; Rand-2 OMe,

RNA oligonucleotides and 2 -O-methylated oligonucleotides were synthesized by. 5 AGACACAAACACCAUUGUCACACUCCACAGC; Rand-2 OMe, Materials and methods Oligonucleotides and DNA constructs RNA oligonucleotides and 2 -O-methylated oligonucleotides were synthesized by Dharmacon Inc. (Lafayette, CO). The sequences were: 122-2 OMe, 5

More information

/04/$15.00/0 Molecular Endocrinology 18(3): Copyright 2004 by The Endocrine Society doi: /me

/04/$15.00/0 Molecular Endocrinology 18(3): Copyright 2004 by The Endocrine Society doi: /me 0888-8809/04/$15.00/0 Molecular Endocrinology 18(3):588 605 Printed in U.S.A. Copyright 2004 by The Endocrine Society doi: 10.1210/me.2003-0090 Increased Cytochrome P450 17 -Hydroxylase Promoter Function

More information

SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION Dynamic Phosphorylation of HP1 Regulates Mitotic Progression in Human Cells Supplementary Figures Supplementary Figure 1. NDR1 interacts with HP1. (a) Immunoprecipitation using

More information

Supplementary Figure S1. Immunodetection of full-length XA21 and the XA21 C-terminal cleavage product.

Supplementary Figure S1. Immunodetection of full-length XA21 and the XA21 C-terminal cleavage product. Supplementary Information Supplementary Figure S1. Immunodetection of full-length XA21 and the XA21 C-terminal cleavage product. Total protein extracted from Kitaake wild type and rice plants carrying

More information

Supplementary Figure 1. Isolation of GFPHigh cells.

Supplementary Figure 1. Isolation of GFPHigh cells. Supplementary Figure 1. Isolation of GFP High cells. (A) Schematic diagram of cell isolation based on Wnt signaling activity. Colorectal cancer (CRC) cell lines were stably transduced with lentivirus encoding

More information

Coleman et al., Supplementary Figure 1

Coleman et al., Supplementary Figure 1 Coleman et al., Supplementary Figure 1 BrdU Merge G1 Early S Mid S Supplementary Figure 1. Sequential destruction of CRL4 Cdt2 targets during the G1/S transition. HCT116 cells were synchronized by sequential

More information

Supplemental figures Supplemental Figure 1: Fluorescence recovery for FRAP experiments depicted in Figure 1.

Supplemental figures Supplemental Figure 1: Fluorescence recovery for FRAP experiments depicted in Figure 1. Supplemental figures Supplemental Figure 1: Fluorescence recovery for FRAP experiments depicted in Figure 1. Percent of original fluorescence was plotted as a function of time following photobleaching

More information

Supplemental Data. Steiner et al. Plant Cell. (2012) /tpc

Supplemental Data. Steiner et al. Plant Cell. (2012) /tpc Supplemental Figure 1. SPY does not interact with free GST. Invitro pull-down assay using E. coli-expressed MBP-SPY and GST, GST-TCP14 and GST-TCP15. MBP-SPY was used as bait and incubated with equal amount

More information

IKK is a therapeutic target in KRAS-induced lung cancer with disrupted p53 activity

IKK is a therapeutic target in KRAS-induced lung cancer with disrupted p53 activity IKK is a therapeutic target in KRAS-induced lung cancer with disrupted p5 activity H6 5 5 H58 A59 H6 H58 A59 anti-ikkα anti-ikkβ anti-panras anti-gapdh anti-ikkα anti-ikkβ anti-panras anti-gapdh anti-ikkα

More information

Supplementary Figure 1: sgrna library generation and the length of sgrnas for the functional screen. (a) A diagram of the retroviral vector for sgrna

Supplementary Figure 1: sgrna library generation and the length of sgrnas for the functional screen. (a) A diagram of the retroviral vector for sgrna Supplementary Figure 1: sgrna library generation and the length of sgrnas for the functional screen. (a) A diagram of the retroviral vector for sgrna expression. It contains a U6-promoter-driven sgrna

More information

Nature Immunology: doi: /ni Supplementary Figure 1. Zranb1 gene targeting.

Nature Immunology: doi: /ni Supplementary Figure 1. Zranb1 gene targeting. Supplementary Figure 1 Zranb1 gene targeting. (a) Schematic picture of Zranb1 gene targeting using an FRT-LoxP vector, showing the first 6 exons of Zranb1 gene (exons 7-9 are not shown). Targeted mice

More information

Parthanatos mediates AIMP2-activated age-dependent dopaminergic neuronal loss

Parthanatos mediates AIMP2-activated age-dependent dopaminergic neuronal loss SUPPLEMENTARY INFORMATION Parthanatos mediates AIMP2-activated age-dependent dopaminergic neuronal loss Yunjong Lee, Senthilkumar S. Karuppagounder, Joo-Ho Shin, Yun-Il Lee, Han Seok Ko, Debbie Swing,

More information

Lecture 25 (11/15/17)

Lecture 25 (11/15/17) Lecture 25 (11/15/17) Reading: Ch9; 328-332 Ch25; 990-995, 1005-1012 Problems: Ch9 (study-guide: applying); 1,2 Ch9 (study-guide: facts); 7,8 Ch25 (text); 1-3,5-7,9,10,13-15 Ch25 (study-guide: applying);

More information

FEBRUARY 12, 2010 VOLUME 285 NUMBER 7 JOURNAL OF BIOLOGICAL CHEMISTRY 4847

FEBRUARY 12, 2010 VOLUME 285 NUMBER 7 JOURNAL OF BIOLOGICAL CHEMISTRY 4847 THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 7, pp. 4847 4858, February 12, 2010 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. The Tumor Suppressor

More information

7.06 Problem Set #3, Spring 2005

7.06 Problem Set #3, Spring 2005 7.06 Problem Set #3, Spring 2005 1. The Drosophila compound eye is composed of about 800 units called ommatidia. Each ommatidium contains eight photoreceptor neurons (R1 through R8), which develop in a

More information

Supplemental Information. HEXIM1 and NEAT1 Long Non-coding RNA Form. a Multi-subunit Complex that Regulates. DNA-Mediated Innate Immune Response

Supplemental Information. HEXIM1 and NEAT1 Long Non-coding RNA Form. a Multi-subunit Complex that Regulates. DNA-Mediated Innate Immune Response Molecular Cell, Volume 67 Supplemental Information HEXIM1 and NEAT1 Long Non-coding RNA Form a Multi-subunit Complex that Regulates DNA-Mediated Innate Immune Response Mehdi Morchikh, Alexandra Cribier,

More information

Supplementary Materials

Supplementary Materials Supplementary Materials Construction of Synthetic Nucleoli in Human Cells Reveals How a Major Functional Nuclear Domain is Formed and Propagated Through Cell Divisision Authors: Alice Grob, Christine Colleran

More information

ENCODE RBP Antibody Characterization Guidelines

ENCODE RBP Antibody Characterization Guidelines ENCODE RBP Antibody Characterization Guidelines Approved on November 18, 2016 Background An integral part of the ENCODE Project is to characterize the antibodies used in the experiments. This document

More information

Supplemental Table 1 Gene Symbol FDR corrected p-value PLOD1 CSRP2 PFKP ADFP ADM C10orf10 GPI LOX PLEKHA2 WIPF1

Supplemental Table 1 Gene Symbol FDR corrected p-value PLOD1 CSRP2 PFKP ADFP ADM C10orf10 GPI LOX PLEKHA2 WIPF1 Supplemental Table 1 Gene Symbol FDR corrected p-value PLOD1 4.52E-18 PDK1 6.77E-18 CSRP2 4.42E-17 PFKP 1.23E-14 MSH2 3.79E-13 NARF_A 5.56E-13 ADFP 5.56E-13 FAM13A1 1.56E-12 FAM29A_A 1.22E-11 CA9 1.54E-11

More information

GUGAUAAUGGAGCGAGAUUUUCUGUUGUGCUUGAUCUAACCAUGUGCUUGCGAGGUAUGA GAAAAACAUGGUUCCGUCAAGCACCAUGGAACGUCACGCAGCUUUCUACA

GUGAUAAUGGAGCGAGAUUUUCUGUUGUGCUUGAUCUAACCAUGUGCUUGCGAGGUAUGA GAAAAACAUGGUUCCGUCAAGCACCAUGGAACGUCACGCAGCUUUCUACA Precursor mmu-mir-8- GUGAUAAUGGAGCGAGAUUUUCUGUUGUGCUUGAUCUAACCAUGUGCUUGCGAGGUAUGA GAAAAACAUGGUUCCGUCAAGCACCAUGGAACGUCACGCAGCUUUCUACA Precursor mmu-mir-8- GACCAGUUGCCGCGGGGCUUUCCUUUGUGCUUGAUCUAACCAUGUGGUGGAACGAUGGAA

More information

Tandem E2F Binding Sites in the Promoter of the p107 Cell Cycle Regulator Control p107 Expression and Its Cellular Functions

Tandem E2F Binding Sites in the Promoter of the p107 Cell Cycle Regulator Control p107 Expression and Its Cellular Functions Tandem E2F Binding Sites in the Promoter of the p107 Cell Cycle Regulator Control p107 Expression and Its Cellular Functions Deborah L. Burkhart 1,2, Stacey E. Wirt 1,2, Anne-Flore Zmoos 1, Michael S.

More information

The microtubule-associated tau protein has intrinsic acetyltransferase activity. Todd J. Cohen, Dave Friedmann, Andrew W. Hwang, Ronen Marmorstein and

The microtubule-associated tau protein has intrinsic acetyltransferase activity. Todd J. Cohen, Dave Friedmann, Andrew W. Hwang, Ronen Marmorstein and SUPPLEMENTARY INFORMATION: The microtubule-associated tau protein has intrinsic acetyltransferase activity Todd J. Cohen, Dave Friedmann, Andrew W. Hwang, Ronen Marmorstein and Virginia M.Y. Lee Cohen

More information

Supplementary Information

Supplementary Information Supplementary Information MLL histone methylases regulate expression of HDLR- in presence of estrogen and control plasma cholesterol in vivo Khairul I. Ansari 1, Sahba Kasiri 1, Imran Hussain 1, Samara

More information

Functional Genomics Research Stream. Research Meeting: June 19, 2012 SYBR Green qpcr, Research Update

Functional Genomics Research Stream. Research Meeting: June 19, 2012 SYBR Green qpcr, Research Update Functional Genomics Research Stream Research Meeting: June 19, 2012 SYBR Green qpcr, Research Update Updates Alternate Lab Meeting Fridays 11:30-1:00 WEL 4.224 Welcome to attend either one Lab Log thanks

More information

Supplementary Figures Montero et al._supplementary Figure 1

Supplementary Figures Montero et al._supplementary Figure 1 Montero et al_suppl. Info 1 Supplementary Figures Montero et al._supplementary Figure 1 Montero et al_suppl. Info 2 Supplementary Figure 1. Transcripts arising from the structurally conserved subtelomeres

More information

Supplemental Data. Lee et al. Plant Cell. (2010) /tpc Supplemental Figure 1. Protein and Gene Structures of DWA1 and DWA2.

Supplemental Data. Lee et al. Plant Cell. (2010) /tpc Supplemental Figure 1. Protein and Gene Structures of DWA1 and DWA2. Supplemental Figure 1. Protein and Gene Structures of DWA1 and DWA2. (A) Protein structures of DWA1 and DWA2. WD40 region was determined based on the NCBI conserved domain databases (B, C) Schematic representation

More information

CHAPTER 3 DEVELOPMENT OF DENV GROUP SPECIFIC REAL TIME RT-PCR

CHAPTER 3 DEVELOPMENT OF DENV GROUP SPECIFIC REAL TIME RT-PCR CHAPTER 3 DEVELOPMENT OF DENV GROUP SPECIFIC REAL TIME RT-PCR 28 Dengue is diagnosed by either detecting virus or antibody to the virus in blood. Isolation of virus in cell culture or in infant mouse brain

More information

MicroRNA Analysis Paired with Novel Cell Health Assays: A Complete Workflow

MicroRNA Analysis Paired with Novel Cell Health Assays: A Complete Workflow MicroRNA Analysis Paired with Novel Cell Health Assays: A Complete Workflow Brad Hook, Ph.D Manager, NA Scientific Applications 2016 Presentation Outline From cells to RNA a seemingly easy, yet complex

More information

Supplementary Fig. 1 related to Fig. 1 Clinical relevance of lncrna candidate

Supplementary Fig. 1 related to Fig. 1 Clinical relevance of lncrna candidate Supplementary Figure Legends Supplementary Fig. 1 related to Fig. 1 Clinical relevance of lncrna candidate BC041951 in gastric cancer. (A) The flow chart for selected candidate lncrnas in 660 up-regulated

More information

Molecular Cell Biology - Problem Drill 11: Recombinant DNA

Molecular Cell Biology - Problem Drill 11: Recombinant DNA Molecular Cell Biology - Problem Drill 11: Recombinant DNA Question No. 1 of 10 1. Which of the following statements about the sources of DNA used for molecular cloning is correct? Question #1 (A) cdna

More information

A novel two-step genome editing strategy with CRISPR-Cas9 provides new insights into telomerase action and TERT gene expression

A novel two-step genome editing strategy with CRISPR-Cas9 provides new insights into telomerase action and TERT gene expression Xi et al. Genome Biology (2015) 16:231 DOI 10.1186/s13059-015-0791-1 RESEARCH A novel two-step genome editing strategy with CRISPR-Cas9 provides new insights into telomerase action and TERT gene expression

More information

Table S1. Primer sequences

Table S1. Primer sequences Table S1. Primer sequences Primers for quantitative PCR Tgf 1 Forward Tgf 1 Reverse Tgf 2Forward Tgf 2Reverse Tgf 3 Forward Tgf 3 Reverse Tgf r1 Forward Tgf r1 Reverse Tgf r2 Forward Tgf r2 Reverse Thbs1

More information

Please purchase PDFcamp Printer on to remove this watermark. DNA microarray

Please purchase PDFcamp Printer on  to remove this watermark. DNA microarray DNA microarray Example of an approximately 40,000 probe spotted oligo microarray with enlarged inset to show detail. A DNA microarray is a multiplex technology used in molecular biology. It consists of

More information

Hossain_Supplemental Figure 1

Hossain_Supplemental Figure 1 Hossain_Supplemental Figure 1 GFP-PACT GFP-PACT Motif I GFP-PACT Motif II A. MG132 (1µM) GFP Tubulin GFP-PACT Pericentrin GFP-PACT GFP-PACT Pericentrin Fig. S1. Expression and localization of Orc1 PACT

More information

Supplementary Figure 1: MYCER protein expressed from the transgene can enhance

Supplementary Figure 1: MYCER protein expressed from the transgene can enhance Relative luciferase activity Relative luciferase activity MYC is a critical target FBXW7 MYC Supplementary is a critical Figures target 1-7. FBXW7 Supplementary Material A E-box sequences 1 2 3 4 5 6 HSV-TK

More information

Supplemental Information. Loss of MicroRNA-7 Regulation Leads. to a-synuclein Accumulation and. Dopaminergic Neuronal Loss In Vivo

Supplemental Information. Loss of MicroRNA-7 Regulation Leads. to a-synuclein Accumulation and. Dopaminergic Neuronal Loss In Vivo YMTHE, Volume 25 Supplemental Information Loss of MicroRNA-7 Regulation Leads to a-synuclein Accumulation and Dopaminergic Neuronal Loss In Vivo Kirsty J. McMillan, Tracey K. Murray, Nora Bengoa-Vergniory,

More information

2. Outline the levels of DNA packing in the eukaryotic nucleus below next to the diagram provided.

2. Outline the levels of DNA packing in the eukaryotic nucleus below next to the diagram provided. AP Biology Reading Packet 6- Molecular Genetics Part 2 Name Chapter 19: Eukaryotic Genomes 1. Define the following terms: a. Euchromatin b. Heterochromatin c. Nucleosome 2. Outline the levels of DNA packing

More information

SureSilencing sirna Array Technology Overview

SureSilencing sirna Array Technology Overview SureSilencing sirna Array Technology Overview Pathway-Focused sirna-based RNA Interference Topics to be Covered Who is SuperArray? Brief Introduction to RNA Interference Challenges Facing RNA Interference

More information

Lecture #1. Introduction to microarray technology

Lecture #1. Introduction to microarray technology Lecture #1 Introduction to microarray technology Outline General purpose Microarray assay concept Basic microarray experimental process cdna/two channel arrays Oligonucleotide arrays Exon arrays Comparing

More information

A 5 P degradation hot spot influences molecular farming of anticancerogenic nuclease TBN1 in tobacco cells

A 5 P degradation hot spot influences molecular farming of anticancerogenic nuclease TBN1 in tobacco cells Plant Cell, Tissue and Organ Culture (PCTOC) A 5 P degradation hot spot influences molecular farming of anticancerogenic nuclease TBN1 in tobacco cells Anna Týcová a,b, Rajen J. J. Piernikarczyk c, Michael

More information

a Award Number: W81XWH TITLE:

a Award Number: W81XWH TITLE: AD Award Number: W81XWH-04-1-0138 TITLE: Imaging Metastatic Prostate Cancer After Genetic Manipulation of Transcriptional Memory Regulators EZH2 and EED PRINCIPAL INVESTIGATOR: Lily Wu, M.D., Ph.D. CONTRACTING

More information

Cambridge University Press

Cambridge University Press Figure 1.1. Model of RNAi pathway in C. elegans. Transmembrane protein SID-1 allows dsrna to enter the cell. In the cytoplasm,dsrna gets processed by DCR-1,existing in a complex with RDE-4,RDE-1 and DRH-1.

More information

Supplemental Information

Supplemental Information Supplemental Information Itemized List Materials and Methods, Related to Supplemental Figures S5A-C and S6. Supplemental Figure S1, Related to Figures 1 and 2. Supplemental Figure S2, Related to Figure

More information

RNA Interference and the World of Small RNAs

RNA Interference and the World of Small RNAs RNA Interference and the World of Small RNAs O, I die, Horatio; The potent poison quite o'er-crows my spirit: I cannot live to hear the news from England; But I do prophesy the election lights On Fortinbras:

More information

Gene Expression Technology

Gene Expression Technology Gene Expression Technology Bing Zhang Department of Biomedical Informatics Vanderbilt University bing.zhang@vanderbilt.edu Gene expression Gene expression is the process by which information from a gene

More information

GTTCGGGTTCC TTTTGAGCAG

GTTCGGGTTCC TTTTGAGCAG Supplementary Figures Splice variants of the SIP1 transcripts play a role in nodule organogenesis in Lotus japonicus. Wang C, Zhu H, Jin L, Chen T, Wang L, Kang H, Hong Z, Zhang Z. 5 UTR CDS 3 UTR TCTCAACCATCCTTTGTCTGCTTCCGCCGCATGGGTGAGGTCATTTTGTCTAGATGACGTGCAATTTACAATGA

More information

MISSION shrna Library: Next Generation RNA Interference

MISSION shrna Library: Next Generation RNA Interference Page 1 of 6 Page 1 of 6 Return to Web Version MISSION shrna Library: Next Generation RNA Interference By: Stephanie Uder, Henry George, Betsy Boedeker, LSI Volume 6 Article 2 Introduction The technology

More information

microrna-122 stimulates translation of Hepatitis C Virus RNA

microrna-122 stimulates translation of Hepatitis C Virus RNA Supplementary information for: microrna-122 stimulates translation of Hepatitis C Virus RNA Jura Inga Henke 1,5, Dagmar Goergen 1,5, Junfeng Zheng 3, Yutong Song 4, Christian G. Schüttler 2, Carmen Fehr

More information

Problem Set 8. Answer Key

Problem Set 8. Answer Key MCB 102 University of California, Berkeley August 11, 2009 Isabelle Philipp Online Document Problem Set 8 Answer Key 1. The Genetic Code (a) Are all amino acids encoded by the same number of codons? no

More information

Supplementary Figure 1: Expression of RNF8, HERC2 and NEURL4 in the cerebellum and knockdown of RNF8 by RNAi (a) Lysates of the cerebellum from rat

Supplementary Figure 1: Expression of RNF8, HERC2 and NEURL4 in the cerebellum and knockdown of RNF8 by RNAi (a) Lysates of the cerebellum from rat Supplementary Figure 1: Expression of RNF8, HERC2 and NEURL4 in the cerebellum and knockdown of RNF8 by RNAi (a) Lysates of the cerebellum from rat pups at P6, P14, P22, P30 and adult (A) rats were subjected

More information

TOOLS sirna and mirna. User guide

TOOLS sirna and mirna. User guide TOOLS sirna and mirna User guide Introduction RNA interference (RNAi) is a powerful tool for suppression gene expression by causing the destruction of specific mrna molecules. Small Interfering RNAs (sirnas)

More information

Supplemental Information. Boundary Formation through a Direct. Threshold-Based Readout. of Mobile Small RNA Gradients

Supplemental Information. Boundary Formation through a Direct. Threshold-Based Readout. of Mobile Small RNA Gradients Developmental Cell, Volume 43 Supplemental Information Boundary Formation through a Direct Threshold-Based Readout of Mobile Small RNA Gradients Damianos S. Skopelitis, Anna H. Benkovics, Aman Y. Husbands,

More information

Journal of Cell Science Supplementary Material

Journal of Cell Science Supplementary Material 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 SUPPLEMENTARY FIGURE LEGENDS Figure S1: Eps8 is localized at focal adhesions and binds directly to FAK (A) Focal

More information

CHAPTER 9 DNA Technologies

CHAPTER 9 DNA Technologies CHAPTER 9 DNA Technologies Recombinant DNA Artificially created DNA that combines sequences that do not occur together in the nature Basis of much of the modern molecular biology Molecular cloning of genes

More information

Supporting Information

Supporting Information Supporting Information Horie et al. 10.1073/pnas.1008499107 SI Materials and Methods ell ulture and Reagents. THP-1 cells were obtained from the American Type ell ollection. THP-1 cells were transformed

More information

ADAR1 Forms a Complex with Dicer to Promote MicroRNA Processing and RNA-Induced Gene Silencing

ADAR1 Forms a Complex with Dicer to Promote MicroRNA Processing and RNA-Induced Gene Silencing ADAR1 Forms a Complex with Dicer to Promote MicroRNA Processing and RNA-Induced Gene Silencing Hiromitsu Ota, 1,2 Masayuki Sakurai, 1,2 Ravi Gupta, 1,2 Louis Valente, 1 Bjorn-Erik Wulff, 1 Kentaro Ariyoshi,

More information

Familial Cancer Predisposition Syndromes. Retinoblastoma (RB) 13q14 Familial Adenomatous Polyposis (APC) 5q21

Familial Cancer Predisposition Syndromes. Retinoblastoma (RB) 13q14 Familial Adenomatous Polyposis (APC) 5q21 Familial Cancer Predisposition Syndromes Retinoblastoma (RB) 13q14 Familial Adenomatous Polyposis (APC) 5q21 Retinoblastoma Eye Retinal Epithelium develops in the first five years of life Retinoblastoma

More information

Nature Methods: doi: /nmeth Supplementary Figure 1. Validation of RaPID with EDEN15

Nature Methods: doi: /nmeth Supplementary Figure 1. Validation of RaPID with EDEN15 Supplementary Figure 1 Validation of RaPID with EDEN15 (a) Full Western Blot of conventional biotinylated RNA pulldown with EDEN15 and scrambled control (n=3 biologically independent experiments, representative

More information

Microarrays: since we use probes we obviously must know the sequences we are looking at!

Microarrays: since we use probes we obviously must know the sequences we are looking at! These background are needed: 1. - Basic Molecular Biology & Genetics DNA replication Transcription Post-transcriptional RNA processing Translation Post-translational protein modification Gene expression

More information

The lineage-defining factors T-bet and Bcl-6 collaborate to regulate Th1 gene expression patterns

The lineage-defining factors T-bet and Bcl-6 collaborate to regulate Th1 gene expression patterns The lineage-defining factors T-bet and Bcl-6 collaborate to regulate Th1 gene expression patterns Kenneth J. Oestreich, 1 Albert C. Huang, 1,2 and Amy S. Weinmann 1,2 1 Department of Immunology and 2 Molecular

More information

SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION Legends for Supplementary Tables. Supplementary Table 1. An excel file containing primary screen data. Worksheet 1, Normalized quantification data from a duplicated screen: valid

More information

mmu-mir-200a-3p Real-time RT-PCR Detection and U6 Calibration Kit User Manual MyBioSource.com Catalog # MBS826230

mmu-mir-200a-3p Real-time RT-PCR Detection and U6 Calibration Kit User Manual MyBioSource.com Catalog # MBS826230 mmu-mir-200a-3p Real-time RT-PCR Detection and U6 Calibration Kit User Manual Catalog # MBS826230 For the detection and quantification of mirnas mmu-mir-200a-3p normalized by U6 snrna using Real-time RT-PCR

More information

Fig. S1. Nature Medicine: doi: /nm HoxA9 expression levels BM MOZ-TIF2 AML BM. Sca-1-H. c-kit-h CSF1R-H CD16/32-H. Mac1-H.

Fig. S1. Nature Medicine: doi: /nm HoxA9 expression levels BM MOZ-TIF2 AML BM. Sca-1-H. c-kit-h CSF1R-H CD16/32-H. Mac1-H. A 1 4 1 4 1 4 CSF1RH 1 3 1 2 1 1 CSF1RH 1 3 1 2 1 1 CSF1RH 1 3 1 2 1 1 1 1 1 1 1 2 1 3 1 4 GFPH 1 1 1 1 1 2 1 3 1 4 Sca1H 1 1 1 1 1 2 1 3 1 4 ckith 1 4 1 4 1 4 CSF1RH 1 3 1 2 1 1 CSF1RH 1 3 1 2 1 1 CSF1RH

More information

Supplemental Data. LMO4 Controls the Balance between Excitatory. and Inhibitory Spinal V2 Interneurons

Supplemental Data. LMO4 Controls the Balance between Excitatory. and Inhibitory Spinal V2 Interneurons Neuron, Volume 61 Supplemental Data LMO4 Controls the Balance between Excitatory and Inhibitory Spinal V2 Interneurons Kaumudi Joshi, Seunghee Lee, Bora Lee, Jae W. Lee, and Soo-Kyung Lee Supplemental

More information

Correction: The Leukemia-Associated Mllt10/ Af10-Dot1l Are Tcf4/β-Catenin Coactivators Essential for Intestinal Homeostasis

Correction: The Leukemia-Associated Mllt10/ Af10-Dot1l Are Tcf4/β-Catenin Coactivators Essential for Intestinal Homeostasis CORRECTION Correction: The Leukemia-Associated Mllt10/ Af10-Dot1l Are Tcf4/β-Catenin Coactivators Essential for Intestinal Homeostasis Tokameh Mahmoudi, Sylvia F. Boj, Pantelis Hatzis, Vivian S. W. Li,

More information

RNA-Guided Gene Activation by CRISPR-Cas9-Based Transcription Factors

RNA-Guided Gene Activation by CRISPR-Cas9-Based Transcription Factors Supplementary Information RNA-Guided Gene Activation by CRISPR-Cas9-Based Transcription Factors Pablo Perez-Pinera 1, Daniel D. Kocak 1, Christopher M. Vockley 2,3, Andrew F. Adler 1, Ami M. Kabadi 1,

More information

Nature Structural and Molecular Biology: doi: /nsmb.2959

Nature Structural and Molecular Biology: doi: /nsmb.2959 Supplementary Figure 1 EIciNAs were pulled down with an antibody to Pol II. (a) Western blot showing that pol II was efficiently pulled down with a pol II antibody in HeLa cell lysates. (b) The enrichment

More information

Figure S1: Insert sequences for GR1, GR2, Qc3c_AgeI and GR3 vectors

Figure S1: Insert sequences for GR1, GR2, Qc3c_AgeI and GR3 vectors Figure S1: Insert sequences for GR1, GR2, Qc3c_AgeI and GR3 vectors A Vector GR1 SacI BamHI CTCTGGCTAACTAGGC Insert 5/7nt - G TCGAGAGACCGATTGATCCG Insert 5/7nt - CCTAG 1 G CAACAACAACAACAACAACAACAACAACAACAACAACAACAACAACAACAACAACAACAACAAC

More information

Figure S2. Response of mouse ES cells to GSK3 inhibition. Mentioned in discussion

Figure S2. Response of mouse ES cells to GSK3 inhibition. Mentioned in discussion Stem Cell Reports, Volume 1 Supplemental Information Robust Self-Renewal of Rat Embryonic Stem Cells Requires Fine-Tuning of Glycogen Synthase Kinase-3 Inhibition Yaoyao Chen, Kathryn Blair, and Austin

More information

SOD1 as a Molecular Switch for Initiating the Homeostatic ER Stress Response under Zinc Deficiency

SOD1 as a Molecular Switch for Initiating the Homeostatic ER Stress Response under Zinc Deficiency Molecular Cell, Volume 52 Supplemental Information SOD1 as a Molecular Switch for Initiating the Homeostatic ER Stress Response under Zinc Deficiency Kengo Homma, Takao Fujisawa, Naomi Tsuburaya, Namiko

More information

Technical Review. Real time PCR

Technical Review. Real time PCR Technical Review Real time PCR Normal PCR: Analyze with agarose gel Normal PCR vs Real time PCR Real-time PCR, also known as quantitative PCR (qpcr) or kinetic PCR Key feature: Used to amplify and simultaneously

More information

MIT Department of Biology 7.013: Introductory Biology - Spring 2005 Instructors: Professor Hazel Sive, Professor Tyler Jacks, Dr.

MIT Department of Biology 7.013: Introductory Biology - Spring 2005 Instructors: Professor Hazel Sive, Professor Tyler Jacks, Dr. MIT Department of Biology 7.01: Introductory Biology - Spring 2005 Instructors: Professor Hazel Sive, Professor Tyler Jacks, Dr. Claudette Gardel iv) Would Xba I be useful for cloning? Why or why not?

More information

Bacterial DNA replication

Bacterial DNA replication Bacterial DNA replication Summary: What problems do these proteins solve? Tyr OH attacks PO4 and forms a covalent intermediate Structural changes in the protein open the gap by 20 Å! 1 Summary: What problems

More information

Supplementary Materials

Supplementary Materials Supplementary Materials Supplementary Methods Supplementary Discussion Supplementary Figure 1 Calculated frequencies of embryo cells bearing bi-allelic alterations. Targeted indel mutations induced by

More information

Supporting Online Material for

Supporting Online Material for www.sciencemag.org/cgi/content/full/323/5910/124/dc1 Supporting Online Material for Regulation of Neuronal Survival Factor MEF2D by Chaperone-Mediated Autophagy Qian Yang, Hua She, Marla Gearing, Emanuela

More information