Cell proliferation was measured with Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan).
|
|
- Dina Newman
- 5 years ago
- Views:
Transcription
1 Supplemental Materials and Methods Cell proliferation assay Cell proliferation was measured with Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan). GCs were plated at 96-well plates at approximately x 10 4 cells per well and cultured in the growth medium. After 48h transfection, each well were added with 10 μl of the Cell Counting Kit-8 solution and then incubated at 37 ºC for 2 h. Cell number assays were performed in a 96-well format plate reader (ELX 800 universal microplate Reader; BioTek, Inc., Highland Park, IL) by measuring the absorbance at a wavelength of 450 nm (OD450) Supplemental Figures 1
2 21 2
3 Supplemental Fig. S1. Expression and function of mir-383 in GCs. A, mir-383 expression levels in mouse ovarian preantral follicles. Total RNA was extracted from the follicles with different diameters and then subjected to real-time PCR. Data obtained from the follicles with μm in diameter were arbitrarily set at 1.0. B, Hematoxylin and eosin (HE) staining of mouse ovarian tissues collected at 0, 4, 12, 24 and 48h post PMSG injection. C, Serum E 2 levels in PMSG-treated mice at 0, 4, 12, 24 and 48h. D, mir-383 expression levels in the ovaries from PMSG-treated mice at 0, 4, 12, 24 and 48h. E and F, The efficacy of mir-383 mimics or inhibitors was evaluated by real-time PCR. mgcs were transfected with the mir-383 mimics (120nM) (E) or mir-383 inhibitors (150nM) (F) for 48h. The intracellular expression levels of mir-383 were examined by real-time PCR. G, Both mir-383 mimics and inhibitors did not alter GC proliferation. mgcs were transfected with 120 nm each of mir-383 mimics and mimics NC, or 150nM each of mir-383 inhibitors and inhibitors NC for 24, 48 and 72h. Cell viability was measured by the cell counting kit-8 assay after transfection. H, mir-383 did not alter apoptosis of GCs. The apoptosis rate was measured using flow cytometry with Annexin V and PI staining after transfection either with 120 nm each of mir-383 mimics and mimics NC, or 150nM each of mir-383 inhibitors and inhibitors NC for 48h. I and J, mir-383 mimics enhanced Cyp19a1 protein expression, while knockdown of endogenous mir-383 suppressed Cyp19a1 protein expression in GCs. GCs were transfected with oligonucleotides for 48 h, and Cyp19a1 protein levels were determined by Western blotting. Representative Western blot for Cyp19a1 (upper bands) and GAPDH (lower bands) (I) and the corresponding densitometric analysis (J) are shown. The ratio of the Cyp19a1 band intensity over the GAPDH band intensity in controls was arbitrarily set at 1.0. K, Both mir-383 mimics and inhibitors did 42 not alter Cyp11a1 mrna expression levels. mgcs were transfected with oligonucleotides for 48 h. Total 3
4 RNA was extracted from GCs, and the Cyp11a1 mrna levels were determined by real-time PCR analysis. L, mir-383 expression levels in GCs, MEF and RAW164.7 cells. Total RNA was extracted from GCs, MEF and RAW164.7 cells and then subjected to real-time PCR. Data obtained from the MEF was arbitrarily set at 1.0. In A, C-H and J-L, data shown represent the mean ± S.E.M of three independent experiments performed in triplicate. ** P < NC, negative control. 48 4
5 Supplemental Fig. S2. RBMS1 is a target of mir-383. A, Schematic diagram of the putative binding sites for mouse (mmu) mir-383 and human (hsa) mir-383 in the RBMS1 3 UTR. The mutated residues 52 of mir-383 binding sites are shown in red. RBMS1 3 UTR MT, mutation-type RBMS1 3 UTR. B-D, 5
6 Hsa-miR-383 reduces RBMS1 protein (B and C) and mrna (D) expression in MCF-7 cells. MCF-7 cells were transfected with 120 nm each of mir-383 mimics and mimics NC, or 150 nm each of mir-383 inhibitors and inhibitors NC. The expression levels of RBMS1 mrna and protein were determined by real-time PCR and Western blotting analysis, respectively. Representative Western blot for RBMS1 (upper bands) and GAPDH (lower bands) (B) and the corresponding densitometric analysis (C) are shown. The ratio of the RBMS1 band intensity over the GAPDH band intensity in controls was arbitrarily set at 1.0. E, mir-383 inhibits RBMS1 3 UTR luciferase activity. Luciferase reporters containing wild-type RBMS1 3 UTR were cotransfected with 10, 20, 30, 40, 60 nm mir-383 mimics or mimics NC. Luciferase activity was measured 30 h after transfection. F, Efficacy of RBMS1 overexpression and RBMS1 sirna. Protein extracts from GCs transfected with 1μg/ml each of pegfp-c1 or pegfp-rbms1 vector or sirna negative control (si.nc) and RBMS1 sirna (si-rbms1), were immunoblotted with anti-rbms1 antibodies. G, RBMS1 attenuated E 2 release from GCs. GCs were transfected with different concentrations (0.5, 1.0, 1.5μg/ml) of pegfp-c1 and pegfp-rbms1 vector for 48h. After culture, media were collected for measurement of E 2 levels. H-I, Enforced RBMS1 expression and knockdown of RBMS1 (si-rbms1) did not alter GC proliferation and apoptosis. GCs were transfected with EGFP- RBMS1 vector/ control vector pegfp-c1 or si-rbms1/si.nc for 24, 48, 72h. Cell viability was measured by the cell counting kit-8 assay after transfection. The apoptosis rate was measured using flow cytometry with Annexin V and PI staining after transfection either with EGFP- RBMS1 vector/ control vector pegfp-c1 or si-rbms1/si.nc for 48 h. NC, negative control. In C-E and G-I, data shown represent the mean ± S.E.M of three independent experiments performed in triplicate. ** 73 P <
7 Supplemental Fig. S3. c-myc is not a target of mir-383. A and B, mir-383 did not target 3 UTR of c-myc. HEK 293T cells were cotransfected with luciferase reporters containing the c-myc 3 UTR and 10, 20, 30nM of mir-383 mimics/ mimics NC or 30nM of mir-383 inhibitors and inhibitors NC. C and D, Effects of RBMS1 on c-myc protein expression in GCs. c-myc was determined by Western blotting after transfection of si-rbms1/si.nc or pegfp-rbms1 /control into GCs. Representative Western blot for c-myc (upper bands) and GAPDH (lower bands) (C) and the corresponding densitometric analysis (D) are shown. The ratio of the c-myc band intensity over the GAPDH band intensity in controls was arbitrarily 7
8 set at 1.0. E, Efficacy of c-myc overexpression and c-myc sirna. Protein extracts from GCs transfected either with EGFP-c-Myc vector/ control vector pegfp-c1 or si-rbms1/si.nc for 48 h, were immunoblotted with anti-c-myc antibodies. In A, B and D, data shown represent the mean ± S.E.M of three independent experiments performed in triplicate. ** P <
9 87 9
10 Supplemental Fig. S4. SF-1 transactivates mir-383 expression. A, Schematic representation of the mir-383 genomic locus hosted in the intron 1 of SGCZ. B, Overexpression of SF-1 enhances the RNA levels of SGCZ, pri-mir-383, pre-mir-383 and mature mir-383. NIH3T3 cells were transfected with pegfp-rbms1 vector /control vector, and the RNA levels were determined after 48 h transfection by real-time PCR. C, FSH promotes the expression of SGCZ mrna, mir-383 and SF-1 mrna in GCs. mgcs were treated with FSH (100ng/ml) for 6 h and subjected to real-time PCR analysis. D and E, Expression patterns of SF-1 and mir-383 in the ovary. D, Total RNA was extracted from the PND 1, PND 4, PND 9, PND 14, and PND 21 mouse ovaries and then subjected to real-time PCR. The SF-1 mrna and mir-383 levels (D) were normalized to GAPDH and U6 for each sample, respectively. Data obtained from the PND 1 mouse ovary were arbitrarily set at 1.0. E, SF-1 protein expression levels were determined by Western blotting analysis in the PND 1, PND 4, PND 9, PND 14, PND 21 mouse ovaries. Representative Western blot for SF-1 (upper bands) and GAPDH (lower bands) (left panel of Fig. S4E) and the corresponding densitometric analysis (right panel of Fig. S4E) are shown. The ratio of the SF-1 band intensity over the GAPDH band intensity in PND 1 ovaries was arbitrarily set at 1.0. F, SF-1 mrna expression levels in various stages of follicle development: preantral, antral and ovulated. Total RNA was extracted from the different stage follicles and then subjected to real-time PCR. Data obtained from the preantral follicles was arbitrarily set at 1.0. G, Changes in SF-1 mrna expression in ovaries of PMSG/hCG-treated immature mice. Total RNA was extracted from ovaries of PMSG-treated mice at 24 h and PMSG/hCG-treated mice at indicated post-hcg time points, and then subjected to real-time PCR. H, mir-383 had no effect on the expression level of SF-1 protein in GCs. GCs were transfected with 120 nm each of mir-383 mimics and mimics NC, or 150 nm each of mir-383 inhibitors and inhibitors NC. SF-1 protein expression levels were determined by Western blotting analysis. I, Mutational analysis of SGCZ promoter-luciferase reporter constructs. Left panel, schematic representation of 5 - or 3 - mutant fragments of the mouse SGCZ promoter in conjunction with the luciferase gene (LUC) in pgl3-basic vector. The nucleotides are numbered from the transcriptional starting site that was assigned +1. Right panel, luciferase activity of the mutant promoter constructs. SGCZ (-1489/+56), SGCZ (-1489/+56) 10
11 mutant 1 (mutated site at -980 bp) and SGCZ (-1489/+56) mutant 2 (mutated site at -419 bp) promoter constructs were cloned into the luciferase reporter vector pgl3-basic. Relative luciferase activity was calculated as a ratio of luciferase activity of SGCZ mutant constructs driven by SF-1 over the pgl3-basic empty vector, which was arbitrarily set at 1. J, ChIP analysis reveals in vivo binding of SF-1 to the SGCZ promoter. ChIP assays were performed on NIH3T3 cell extracts using antibodies against IgG and SF-1, followed by PCR amplification with primers sequences corresponding to the mouse SGCZ promoter region between nucleotides -200 and -8. In B-G, I, data were presented as means ± S.E.M for at least 3 independent experiments. * P < 0.05, ** P < NC, negative control
12 Supplemental Fig. S5. SF-1 mediates the effects of mir-383/ RBMS1/c-Myc on the Cyp19a1 mrna expression in GCs. A, C and D, SF-1 overexpression in mgcs increased the suppressed Cyp19a1 mrna levels by mir-383 inhibitors (A), overexpression of RBMS1 (C) or of c-myc (D). B, si-sf-1 partially abrogated mir-383-induced Cyp19a1 expression in mgcs. E, mir-383 inhibitors-induced decrease of Cyp19a1 mrna levels in GCs was further suppressed by si-sf-1. GCs were co-transfected with 12
13 pegfp-sf-1 and indicated expression vectors or mir-383 inhibitors, or with si-sf-1 and mir-383 mimics/inhibitors for 48 h. The culture medium was collected for hormone analysis. Data were presented as means ± S.E.M for at least 3 independent experiments. * P < 0.05, ** P < NC, negative control. 13
14 Supplementary Table S1. PCR and RT-PCR primers used in this study Gene Forward primer sequence (5-3 ) RBMS1 GCTAGAATTCTATGGGCAAAGTGTGGAAACAAC c-myc GTACGAATTCTCTGGATTTCCTTTGGGCGTT SF-1 GTATCCTCGAGCTATGGACTATTCGTACGACGAG RBMS1 3'UTR CTACTCTCGAGCTGTGAGATGTACCGAAGGGAGT c-myc 3'UTR GTTATGCGGCCGCACTGACCTAACTCGAGGA SGCZ promoter(-1489/+56) GCATCGGTACCGGCCCACAAATTATGGATTATT SGCZ promoter(-1489/+56) mutant1 TGATATCAGTTCCGCATACCATTTATTGAAGAAG SGCZ promoter(-1489/+56) mutant 2GATGTAGACTGGAGTTAGAATGAATTCAAAGAA SGCZ promoter(-703/+56) GTATCGGTACCAGCTCAGCAGTGCAGAAATATG SGCZ promoter(-524/+56) GTGTCGGTACCACTGAAGCCTTAACATAAATCA SGCZ promoter(-230/+56) GTATCGGTACCTGATACAGGCAAGCCTGTGTCT SGCZ promoter(-150/+56) GTATCGGTACCCCTAGTAGATTCCGGACAGAGG SGCZ promoter(-128/+56) GTATCGGTACCGCAGTCTTTAAGACCCAGCG SGCZ promoter(-100/+56) GTATCGGTACCTGTCTGGAGAGGAAATGTTGCT SGCZ promoter(-128/+56) mutant fogtatcggtaccgctgacataatgtcgctgcc Supplementary Table S2. Sequence of oligonucleotides Name Sequence (5' to 3') Inhibitor NC CAGUACUUUUGUGUAGUACAA mmu-mir-383 inhibitors AGCCACAGUCACCUUCUGAUCU mimics NC UUCUCCGAACGUGUCACGUTT ACGUGACACGUUCGGAGAATT mmu-mir-383 mimics AGAUCAGAAGGUGACUGUGGCU CCACAGUCACCUUCUGAUCUUU LNA scramble-mir GTGTAACACGTCTATACGCCCA LNA mmu-mir-383 AGCCACAGTCACCTTCTGATCT Supplementary Table S3. qrt-pcr primers used in this study Gene Forward primer sequence (5-3 ) Cyp19a1 TGTGTTGACCCTCATGAGACA Cyp11a1 TCCCTGTAAATGGGGCCATAC GAPDH TGTGTCCGTCGTGGATCTGA RBMS1 CTGAGCAAGACAAACCTCTACAT Pri-miR-383 TCGACCACTTCAGTGACTGA Pre-miR-383 CTCAGATCAGAAGGTGACTG c-myc CAGAGGAGGAACGAGCTGAAGCGC FAS GATGCACACTCTGCGATGAAG SGCZ TGAGAGTCACCAAGAAGGGA SF-1 ATGGCCGACCAGACCTTTATCTC
15 Reverse primer sequence (5-3 ) GCATGTCGACTTACTTATTGGGTGGAAAGGTAT CTAGGTCGACTTATGCACCAGAGTTTCGAAG GACATGAATTCTCAAGTCTGCTTGGCCTGCAGC GTGTAGCGGCCGCACTTTACATTGCATCATACAGCAG GCAGCGTTTAAACTTCAAGGCCCTATTTACATGGG GGTATGCGGAACTGATATCAGACCAGTCTTATTT TTCTAACTCCAGTCTACATCGCACTTGTAATCTG Reverse primer sequence (5-3 ) CTTGACGGATCGTTCATACTTTC AGGTCCTTCAATGAGATCCCTT TTGCTGTTGAAGTCGCAGGAG GGCCTTATCCAAAATCGCCTT CTCTTTCTGACCAGGCAGTG CTGACCAGGCAGTGCTGTGG TTATGCACCAGAGTTTCGAAGCTGTTCG CAGTGTTCACAGCCAGGAGAAT GTTCCTTGCATTCACTGTTA TGGTCCAACACCAGCAGCTC
Supplementary Figure 1 Validate the expression of mir-302b after bacterial infection by northern
Supplementary Figure 1 Validate the expression of mir-302b after bacterial infection by northern blot. Northern blot analysis of mir-302b expression following infection with PAO1, PAK and Kp in (A) lung
More informationSUPPLEMENTARY INFORMATION
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.5 3-3. -. b Fold change by treatment ( (+)/(-)) 8
More information8Br-cAMP was purchased from Sigma (St. Louis, MO). Silencer Negative Control sirna #1 and
1 Supplemental information 2 3 Materials and Methods 4 Reagents and animals 5 8Br-cAMP was purchased from Sigma (St. Louis, MO). Silencer Negative Control sirna #1 and 6 Silencer Select Pre-designed sirna
More informationSupplementary Table 1. Sequences for BTG2 and BRCA1 sirnas.
Supplementary Table 1. Sequences for BTG2 and BRCA1 sirnas. Target Gene Non-target / Control BTG2 BRCA1 NFE2L2 Target Sequence ON-TARGET plus Non-targeting sirna # 1 (Cat# D-001810-01-05) sirna1: GAACCGACAUGCUCCCGGA
More informationSupplementary data. sienigma. F-Enigma F-EnigmaSM. a-p53
Supplementary data Supplemental Figure 1 A sienigma #2 sienigma sicontrol a-enigma - + ++ - - - - - - + ++ - - - - - - ++ B sienigma F-Enigma F-EnigmaSM a-flag HLK3 cells - - - + ++ + ++ - + - + + - -
More informationFig. S1. Effect of p120-catenin overexpression on the interaction of SCUBE2 with E-cadherin. The expression plasmid encoding FLAG.
Fig. S1. Effect of p120-catenin overexpression on the interaction of SCUBE2 with E-cadherin. The expression plasmid encoding FLAG.SCUBE2, E-cadherin.Myc, or HA.p120-catenin was transfected in a combination
More informationOnline Supplementary Information
Online Supplementary Information NLRP4 negatively regulates type I interferon signaling by targeting TBK1 for degradation via E3 ubiquitin ligase DTX4 Jun Cui 1,4,6,7, Yinyin Li 1,5,6,7, Liang Zhu 1, Dan
More informationSUPPLEMENTARY INFORMATION
DOI: 10.1038/ncb3240 Supplementary Figure 1 GBM cell lines display similar levels of p100 to p52 processing but respond differentially to TWEAK-induced TERT expression according to TERT promoter mutation
More informationSupplementary Figure 1, related to Figure 1. GAS5 is highly expressed in the cytoplasm of hescs, and positively correlates with pluripotency.
Supplementary Figure 1, related to Figure 1. GAS5 is highly expressed in the cytoplasm of hescs, and positively correlates with pluripotency. (a) Transfection of different concentration of GAS5-overexpressing
More informationTable 1. Primers, annealing temperatures, and product sizes for PCR amplification.
Table 1. Primers, annealing temperatures, and product sizes for PCR amplification. Gene Direction Primer sequence (5 3 ) Annealing Temperature Size (bp) BRCA1 Forward TTGCGGGAGGAAAATGGGTAGTTA 50 o C 292
More informationtranscription and the promoter occupancy of Smad proteins. (A) HepG2 cells were co-transfected with the wwp-luc reporter, and FLAG-tagged FHL1,
Supplementary Data Supplementary Figure Legends Supplementary Figure 1 FHL-mediated TGFβ-responsive reporter transcription and the promoter occupancy of Smad proteins. (A) HepG2 cells were co-transfected
More informationHCT116 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 informationASPP1 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 informationsupplementary information
Figure S1 ZEB1 full length mrna. (a) Analysis of the ZEB1 mrna using the UCSC genome browser (http://genome.ucsc.edu) revealed truncation of the annotated Refseq sequence (NM_030751). The probable terminus
More informationEffects of metformin on Sirt1, Nrf2 and AhR expression in cancer cells
A Dissertation for the Degree of Doctor of Philosophy Effects of metformin on Sirt1, Nrf2 and AhR expression in cancer cells Department of Pharmacy Graduate School Chungnam National University By Minh
More informationSupplementary Figure 1. Expressions of stem cell markers decreased in TRCs on 2D plastic. TRCs were cultured on plastic for 1, 3, 5, or 7 days,
Supplementary Figure 1. Expressions of stem cell markers decreased in TRCs on 2D plastic. TRCs were cultured on plastic for 1, 3, 5, or 7 days, respectively, and their mrnas were quantified by real time
More informationb alternative classical none
Supplementary Figure. 1: Related to Figure.1 a d e b alternative classical none NIK P-IkBa Total IkBa Tubulin P52 (Lighter) P52 (Darker) RelB (Lighter) RelB (Darker) HDAC1 Control-Sh RelB-Sh NF-kB2-Sh
More informationSUPPLEMENTAL MATERIAL
SUPPLEMENTAL MATERIAL MATERIALS AND METHODS Generation of TSPOΔ/Δ murine embryonic fibroblasts Embryos were harvested from 13.5-day pregnant TSPOfl/fl mice. After dissection to eviscerate and remove the
More informationSupplemental Figure 1. HepG2 cells were transfected with GLI luciferase reporter construct
Supplemental Figure 1. HepG2 cells were transfected with GLI luciferase reporter construct (pgl38xgli), EWS-FLI1 luciferase reporter construct (NROB1-Luc) with or without GLI1, EWS- FLI1 and cdnas respectively.
More informationFig Hypoxia causes growth retardation and developmental delay. (A) Morphology of wildtype zebrafish embryos at 48 hours post fertilization
FIGURES AND TABLES Fig. 1-1. Hypoxia causes growth retardation and developmental delay. (A) Morphology of wildtype zebrafish embryos at 48 hours post fertilization (hpf) after 24 h of normoxia or hypoxia
More informationSupplementary Fig. 1 Proteomic analysis of ATR-interacting proteins. ATR, ARID1A and
Supplementary Figure Legend: Supplementary Fig. 1 Proteomic analysis of ATR-interacting proteins. ATR, ARID1A and ATRIP protein peptides identified from our mass spectrum analysis were shown. Supplementary
More informationSupplementary Information. HBx-upregulated lncrna UCA1 promotes cell growth and tumorigenesis
Supplementary Information HBx-upregulated lncrna UCA1 promotes cell growth and tumorigenesis by recruiting EZH2 and repressing p27kip1/cdk2 signaling Jiao-Jiao Hu 1, Wei Song 1, Shao-Dan Zhang 1, Xiao-Hui
More information1. Primers for PCR to amplify hairpin stem-loop precursor mir-145 plus different flanking sequence from human genomic DNA.
Supplemental data: 1. Primers for PCR to amplify hairpin stem-loop precursor mir-145 plus different flanking sequence from human genomic DNA. Strategy#1: 20nt at both sides: #1_BglII-Fd primer : 5 -gga
More informationSupplementary Figure 1
Supplementary Figure 1 Virus infection induces RNF128 expression. (a,b) RT-PCR analysis of Rnf128 (RNF128) mrna expression in mouse peritoneal macrophages (a) and THP-1 cells (b) upon stimulation with
More informationSupplementary information for: Mutant p53 gain-of-function induces epithelial-mesenchymal transition. through modulation of the mir-130b-zeb1 axis
Supplementary information for: Mutant p53 gain-of-function induces epithelial-mesenchymal transition through modulation of the mir-3b-zeb axis AUTHORS: Peixin Dong, Mihriban Karaayvaz, Nan Jia, Masanori
More informationSupplemental Table/Figure Legends
MiR-26a is required for skeletal muscle differentiation and regeneration in mice Bijan K. Dey, Jeffrey Gagan, Zhen Yan #, Anindya Dutta Supplemental Table/Figure Legends Suppl. Table 1: Effect of overexpression
More informationsupplementary information
DOI: 10.1038/ncb2116 Figure S1 CDK phosphorylation of EZH2 in cells. (a) Comparison of candidate CDK phosphorylation sites on EZH2 with known CDK substrates by multiple sequence alignments. (b) CDK1 and
More informationsupplementary 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 informationRegulation of transcription by the MLL2 complex and MLL complex-associated AKAP95
Supplementary Information Regulation of transcription by the complex and MLL complex-associated Hao Jiang, Xiangdong Lu, Miho Shimada, Yali Dou, Zhanyun Tang, and Robert G. Roeder Input HeLa NE IP lot:
More informationIGF-1 promotes the development and cytotoxic activity of human NK cells regulated
Supplementary Information for IGF-1 promotes the development and cytotoxic activity of human NK cells regulated by mir-483-3p Fang Ni 1, 2, Rui Sun 1, Binqing Fu 1, Fuyan Wang 1, Chuang Guo 1, Zhigang
More informationSupplementary Materials for
www.sciencesignaling.org/cgi/content/full/10/496/eaam6291/dc1 Supplementary Materials for Regulation of autophagy, NF-κB signaling, and cell viability by mir-124 in KRAS mutant mesenchymal-like NSCLC cells
More informationDocument S1. Supplemental Experimental Procedures and Three Figures (see next page)
Supplemental Data Document S1. Supplemental Experimental Procedures and Three Figures (see next page) Table S1. List of Candidate Genes Identified from the Screen. Candidate genes, corresponding dsrnas
More informationSupplementary Figure 1. NORAD expression in mouse (A) and dog (B). The black boxes indicate the position of the regions alignable to the 12 repeat
Supplementary Figure 1. NORAD expression in mouse (A) and dog (B). The black boxes indicate the position of the regions alignable to the 12 repeat units in the human genome. Annotated transposable elements
More informationGenomic DNA fragments identified by ChIP-chip assay for MR [28] corresponding to two
Supplemental Materials and Methods Genomic DNA fragments identified by ChIP-chip assay for MR [28] corresponding to two upstream regions of the human Klf9 gene (-5139 to -5771 bp and -3875 to -4211 bp)
More informationTo generate the luciferase fusion to the human 3 UTRs, we sub-cloned the 3 UTR
Plasmids To generate the luciferase fusion to the human 3 UTRs, we sub-cloned the 3 UTR fragments downstream of firefly luciferase (luc) in pgl3 control (Promega). pgl3- CDK6 was made by amplifying a 2,886
More informationSupplementary 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 informationSupplementary Figure 1
Supplementary Figure 1 Supplementary Fig. 1 shrna mediated knockdown of ZRSR2 in K562 and 293T cells. (a) ZRSR2 transcript levels in stably transduced K562 cells were determined using qrt-pcr. GAPDH was
More informationFig. S1. eif6 expression in HEK293 transfected with shrna against eif6 or pcmv-eif6 vector.
Fig. S1. eif6 expression in HEK293 transfected with shrna against eif6 or pcmv-eif6 vector. (a) Western blotting analysis and (b) qpcr analysis of eif6 expression in HEK293 T cells transfected with either
More informationSupplementary Figure 1. GST pull-down analysis of the interaction of GST-cIAP1 (A, B), GSTcIAP1
Legends Supplementary Figure 1. GST pull-down analysis of the interaction of GST- (A, B), GST mutants (B) or GST- (C) with indicated proteins. A, B, Cell lysate from untransfected HeLa cells were loaded
More informationMeCP2. MeCP2/α-tubulin. GFP mir1-1 mir132
Conservation Figure S1. Schematic showing 3 UTR (top; thick black line), mir132 MRE (arrow) and nucleotide sequence conservation (vertical black lines; http://genome.ucsc.edu). a GFP mir1-1 mir132 b GFP
More informationComparative Analysis of Argonaute-Dependent Small RNA Pathways in Drosophila
Molecular Cell, Volume 32 Supplemental Data Comparative Analysis of Argonaute-Dependent Small RNA Pathways in Drosophila Rui Zhou, Ikuko Hotta, Ahmet M. Denli, Pengyu Hong, Norbert Perrimon, and Gregory
More informationCell death analysis using the high content bioimager BD PathwayTM 855 instrument (BD
Supplemental information Materials and Methods: Cell lines, reagents and antibodies: Wild type (A3) and caspase-8 -/- (I9.2) Jurkat cells were cultured in RPMI 164 medium (Life Technologies) supplemented
More informationBlimp-1/PRDM1 rabbit monoclonal antibody (C14A4) was purchased from Cell Signaling
1 2 3 4 5 6 7 8 9 10 11 Supplementary Methods Antibodies Blimp-1/PRDM1 rabbit monoclonal antibody (C14A4) was purchased from Cell Signaling (Danvers, MA) and used at 1:1000 to detect the total PRDM1 protein
More informationSupplementary Materials for
advances.sciencemag.org/cgi/content/full/4/9/eaat5401/dc1 Supplementary Materials for GLK-IKKβ signaling induces dimerization and translocation of the AhR-RORγt complex in IL-17A induction and autoimmune
More informationSupplementary Figure 1. Nur77 and leptin-controlled obesity. (A) (B) (C)
Supplementary Figure 1. Nur77 and leptin-controlled obesity. (A) Effect of leptin on body weight and food intake between WT and KO mice at the age of 12 weeks (n=7). Mice were i.c.v. injected with saline
More informationSupplementary Information. A novel human endogenous retroviral protein inhibits cell-cell fusion. Supplementary Figures:
Supplementary Information A novel human endogenous retroviral protein inhibits cell-cell fusion Jun Sugimoto, Makiko Sugimoto, Helene Bernstein, Yoshihiro Jinno and Danny J. Schust Supplementary Figures:
More informationSupplementary Figures
Supplementary Figures Supplementary Figure 1 Experimental schema for the identification of circular RNAs in six normal tissues and seven cancerous tissues. Supplementary Fiure 2 Comparison of human circrnas
More informationThyroid peroxidase gene expression is induced by lipopolysaccharide involving Nuclear Factor (NF)-κB p65 subunit phosphorylation
1 2 3 4 5 SUPPLEMENTAL DATA Thyroid peroxidase gene expression is induced by lipopolysaccharide involving Nuclear Factor (NF)-κB p65 subunit phosphorylation Magalí Nazar, Juan Pablo Nicola, María Laura
More informationSupplementary Figure 1 Phosphorylated tau accumulates in Nrf2 (-/-) mice. Hippocampal tissues obtained from Nrf2 (-/-) (10 months old, 4 male; 2
Supplementary Figure 1 Phosphorylated tau accumulates in Nrf2 (-/-) mice. Hippocampal tissues obtained from Nrf2 (-/-) (10 months old, 4 male; 2 female) or wild-type (5 months old, 1 male; 11 months old,
More informationTE5 KYSE510 TE7 KYSE70 KYSE140
TE5 KYSE5 TT KYSE7 KYSE4 Supplementary Figure. Hockey stick plots showing input normalized, rank ordered H3K7ac signals for the candidate SE-associated lncrnas in this study. Rpm Rpm Rpm Chip-seq H3K7ac
More informationSupplementary Fig. 1
a FL (1-2266) NL (1-1190) CL (1191-2266) HA-ICE1: - HA-ICE1: - - - FLAG-ICE2: + + + + FLAG-ELL: + + + + + + IP: anti-ha FLAG-ICE2 HA-ICE1-FL HA-ICE1-NL HA-ICE1-CL FLAG-ICE2 b IP: anti-ha FL (1-2266) NL
More informationSupplementary Information Alternative splicing of CD44 mrna by ESRP1 enhances lung colonization of metastatic cancer cell
Supplementary Information Alternative splicing of CD44 mrna by ESRP1 enhances lung colonization of metastatic cancer cell Supplementary Figures S1-S3 Supplementary Methods Supplementary Figure S1. Identification
More informationa KYSE270-CON KYSE270-Id1
a KYSE27-CON KYSE27- shcon shcon sh b Human Mouse CD31 Relative MVD 3.5 3 2.5 2 1.5 1.5 *** *** c KYSE15 KYSE27 sirna (nm) 5 1 Id2 Id2 sirna 5 1 sirna (nm) 5 1 Id2 sirna 5 1 Id2 [h] (pg per ml) d 3 2 1
More informationTOOLS 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 informationSite-Directed Mutagenesis. Mutations in four Smad4 sites of mouse Gat1 promoter
Supplement Supporting Materials and Methods Site-Directed Mutagenesis. Mutations in four Smad4 sites of mouse Gat1 promoter were independently generated using a two-step PCR method. The Smad4 binding site
More informationT H E J O U R N A L O F C E L L B I O L O G Y
T H E J O U R N A L O F C E L L B I O L O G Y Supplemental material Han et al., http://www.jcb.org/cgi/content/full/jcb.201311007/dc1 Figure S1. SIVA1 interacts with PCNA. (A) HEK293T cells were transiently
More informationSupplementary Methods
Supplementary Methods Reverse transcribed Quantitative PCR. Total RNA was isolated from bone marrow derived macrophages using RNeasy Mini Kit (Qiagen), DNase-treated (Promega RQ1), and reverse transcribed
More informationLi et al., Supplemental Figures
Li et al., Supplemental Figures Fig. S1. Suppressing TGM2 expression with TGM2 sirnas inhibits migration and invasion in A549-TR cells. A, A549-TR cells transfected with negative control sirna (NC sirna)
More informationmir-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 informationSupplemental Material Igreja and Izaurralde 1. CUP promotes deadenylation and inhibits decapping of mrna targets. Catia Igreja and Elisa Izaurralde
Supplemental Material Igreja and Izaurralde 1 CUP promotes deadenylation and inhibits decapping of mrna targets Catia Igreja and Elisa Izaurralde Supplemental Materials and methods Functional assays and
More informationLullaby sirna Transfection Reagent - Results
sirna Transfection Reagent - Results OZ Biosciences is delighted to announce the launching of a new sirna transfection reagent: -sirna. This lipid based transfection reagent is specifically designed for
More informationSUPPLEMENTAL MATERIAL. Carvajal et al. Supplemental Table 1. List of quantitative PCR primers used for cdna analyses and
UPPLEMEAL MATERIAL Carvajal et al. upplemental Table 1. List of quantitative PCR primers used for cdna analyses and chromatin immunoprecipitation assays. Figure 1. DNA damage-induced transcriptional repression
More informationGFP 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 informationSUPPLEMENTARY INFORMATION
(Supplementary Methods and Materials) GST pull-down assay GST-fusion proteins Fe65 365-533, and Fe65 538-700 were expressed in BL21 bacterial cells and purified with glutathione-agarose beads (Sigma).
More informationSupplementary figures
Relative intensity Relative intensity Relative intensity Supplementary figures a None Caffeine None Caffeine c None Caffeine 6 6 6 ISG 6 6 6 UBE1L 6 6 6 UBCH8 6 6 6 EFP 1 1 DOX (h) 1 1 CPT (h) 1 1 UV (h)
More informationFile name: Supplementary Information Description: Supplementary figures and supplementary tables. File name: Peer review file Description:
File name: Supplementary Information Description: Supplementary figures and supplementary tables. File name: Peer review file Description: Supplementary Figure 1. dcas9-mq1 fusion protein induces de novo
More informationTable I. Primers used in quantitative real-time PCR for detecting gene expressions in mouse liver tissues
Table I. Primers used in quantitative real-time PCR for detecting gene expressions in mouse liver tissues Gene name Forward primer 5' to 3' Gene name Reverse primer 5' to 3' CC_F TGCCCTGTTGGGGTTG CC_R
More informationIKK 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 informationSUPPLEMENTARY INFORMATION
DOI: 10.1038/ncb3562 In the format provided by the authors and unedited. Supplementary Figure 1 Glucose deficiency induced FH-ATF2 interaction. In b-m, immunoblotting or immunoprecipitation analyses were
More informationSupplementary
Supplementary information Supplementary Material and Methods Plasmid construction The transposable element vectors for inducible expression of RFP-FUS wt and EGFP-FUS R521C and EGFP-FUS P525L were derived
More informationTranscriptional Regulation of Pro-apoptotic Protein Kinase C-delta: Implications for Oxidative Stress-induced Neuronal Cell Death
SUPPLEMENTAL DATA Transcriptional Regulation of Pro-apoptotic Protein Kinase C-delta: Implications for Oxidative Stress-induced Neuronal Cell Death Huajun Jin 1, Arthi Kanthasamy 1, Vellareddy Anantharam
More informationSUPPLEMENTAL FIGURES AND TABLES
SUPPLEMENTAL FIGURES AND TABLES A B Flag-ALDH1A1 IP: α-ac HEK293T WT 91R 128R 252Q 367R 41/ 419R 435R 495R 412R C Flag-ALDH1A1 NAM IP: HEK293T + + - + D NAM - + + E Relative ALDH1A1 activity 1..8.6.4.2
More informationGalina 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 informationStabilization of the Transcription Factor Foxp3 by the Deubiquitinase USP7 Increases Treg-Cell-Suppressive Capacity
Immunity, Volume 39 Supplemental Information Stabilization of the Transcription Factor Foxp3 by the Deubiquitinase USP7 Increases Treg-Cell-Suppressive Capacity Jorg van Loosdregt, Veerle Fleskens, Juan
More informationLegend for Supplemental Figures and Tables
Legend for Supplemental Figures and Tables Supplemental Fig. 1. Negative regulation of the CYP27B1 promoter in a ligand-dependent manner (A) OK-P cells were transfected with pcdna-trα, pcdna-trβ1 or pcdna3
More informationPartial list of differentially expressed genes from cdna microarray, comparing MUC18-
Supplemental Figure legends Table-1 Partial list of differentially expressed genes from cdna microarray, comparing MUC18- silenced and NT-transduced A375SM cells. Supplemental Figure 1 Effect of MUC-18
More informationEmanuela Tumini, Sonia Barroso, Carmen Pérez Calero and Andrés Aguilera
SUPPLEMENTARY INFORMATION Roles of human POLD1 and POLD3 in genome stability Emanuela Tumini, Sonia Barroso, Carmen Pérez Calero and Andrés Aguilera SUPPLEMENTARY METHODS Cell proliferation After sirna
More informationSUPPLEMENTARY INFORMATION
doi:10.1038/nature09732 Supplementary Figure 1: Depletion of Fbw7 results in elevated Mcl-1 abundance. a, Total thymocytes from 8-wk-old Lck-Cre/Fbw7 +/fl (Control) or Lck-Cre/Fbw7 fl/fl (Fbw7 KO) mice
More informationSupporting 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 informationTranslation of HTT mrna with expanded CAG repeats is regulated by
Supplementary Information Translation of HTT mrna with expanded CAG repeats is regulated by the MID1-PP2A protein complex Sybille Krauß 1,*, Nadine Griesche 1, Ewa Jastrzebska 2,3, Changwei Chen 4, Désiree
More informationNature Biotechnology: doi: /nbt Supplementary Figure 1
Supplementary Figure 1 Schematic and results of screening the combinatorial antibody library for Sox2 replacement activity. A single batch of MEFs were plated and transduced with doxycycline inducible
More informationSupplementary Information for. hnrnp Q mediates a phase-dependent translation-coupled mrna decay of mouse. Period3.
Supplementary Information for hnrnp Q mediates a phase-dependent translation-coupled mrna decay of mouse Period3. Do-Yeon Kim, Eunyee Kwak, Sung-Hoon Kim, Kyung-Ha Lee, Kyung-Chul Woo and Kyong-Tai Kim
More informationSupplementary Figures and Figure legends
Supplementary Figures and Figure legends 3 Supplementary Figure 1. Conditional targeting construct for the murine Satb1 locus with a modified FLEX switch. Schematic of the wild type Satb1 locus; the conditional
More informationOmicsLink shrna Clones guaranteed knockdown even in difficult-to-transfect cells
OmicsLink shrna Clones guaranteed knockdown even in difficult-to-transfect cells OmicsLink shrna clone collections consist of lentiviral, and other mammalian expression vector based small hairpin RNA (shrna)
More informationSupplementary 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 informationKhaled_Fig. S MITF TYROSINASE R²= MITF PDE4D R²= Variance from mean mrna expression
Khaled_Fig. S Variance from mean mrna expression.8 2 MITF.6 TYROSINASE.4 R²=.73.2.8.6.4.2.8.6.4.2.8.6.4.2 MALME3M SKMEL28 UACC257 MITF PDE4D R²=.63 4.5 5 MITF 3.5 4 PDE4B 2.5 3 R²=.2.5 2.5.8.6.4.2.8.6.4.2
More informationSupplemental Table S1. RT-PCR primers used in this study
Supplemental Table S1. RT-PCR primers used in this study -----------------------------------------------------------------------------------------------------------------------------------------------
More informationSUPPLEMENTARY INFORMATION
DOI: 10.1038/ncb3363 Supplementary Figure 1 Several WNTs bind to the extracellular domains of PKD1. (a) HEK293T cells were co-transfected with indicated plasmids. Flag-tagged proteins were immunoprecipiated
More information(a) Immunoblotting to show the migration position of Flag-tagged MAVS
Supplementary Figure 1 Characterization of six MAVS isoforms. (a) Immunoblotting to show the migration position of Flag-tagged MAVS isoforms. HEK293T Mavs -/- cells were transfected with constructs expressing
More informationSupplementary Figure 1 a
3 min PMA 45 min PMA AnnexinV-FITC Supplementary Figure 1 5 min PMA 15 min PMA a 9 min PMA 12 min PMA 5 min FGF7 15 min FGF7 3 min FGF7 6 min FGF7 9 min FGF7 12 min FGF7 5 min control 3 min control 6 min
More informationShort hairpin RNA (shrna) against MMP14. Lentiviral plasmids containing shrna
Supplemental Materials and Methods Short hairpin RNA (shrna) against MMP14. Lentiviral plasmids containing shrna (Mission shrna, Sigma) against mouse MMP14 were transfected into HEK293 cells using FuGene6
More informationSupplementary Figure 1. jmj30-2 and jmj32-1 produce null mutants. (a) Schematic drawing of JMJ30 and JMJ32 genome structure showing regions amplified
Supplementary Figure 1. jmj30-2 and jmj32-1 produce null mutants. (a) Schematic drawing of JMJ30 and JMJ32 genome structure showing regions amplified by primers used for mrna expression analysis. Gray
More informationSupplementary Information
Supplementary Information Sam68 modulates the promoter specificity of NF-κB and mediates expression of CD25 in activated T cells Kai Fu 1, 6, Xin Sun 1, 6, Wenxin Zheng 1, 6, Eric M. Wier 1, Andrea Hodgson
More informationRegulation of hepcidin expression by inflammation-induced activin B
Regulation of hepcidin expression by inflammation-induced activin B Yohei Kanamori, Makoto Sugiyama, Osamu Hashimoto, Masaru Murakami, Tohru Matsui and Masayuki Funaba Supplemental methods Liver cell separation
More informationRegulation 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 informationFigure S1. Specificity of polyclonal anti stabilin-1 and anti stabilin-2 antibodies Lysates of 293T cells transfected with empty vector, mouse
Figure S1. Specificity of polyclonal anti stabilin-1 and anti stabilin-2 antibodies Lysates of 293T cells transfected with empty vector, mouse stabilin-1, or mouse stabilin-2 were immunoblotted using anti
More informationmonoclonal antibody. (a) The specificity of the anti-rhbdd1 monoclonal antibody was examined in
Supplementary information Supplementary figures Supplementary Figure 1 Determination of the s pecificity of in-house anti-rhbdd1 mouse monoclonal antibody. (a) The specificity of the anti-rhbdd1 monoclonal
More informationThis is the author's accepted version of the manuscript.
This is the author's accepted version of the manuscript. The definitive version is published in Nature Communications Online Edition: 2015/4/16 (Japan time), doi:10.1038/ncomms7780. The final version published
More informationSupplementary Methods
Supplementary Methods Microarray Data Analysis Gene expression data were obtained by hybridising a total of 24 samples from 6 experimental groups (n=4 per group) to Illumina HumanHT-12 Expression BeadChips.
More informationSupporting 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