Supplemental Information. Kinesin 1 Drives Autolysosome Tubulation
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1 Developmental Cell, Volume 37 Supplemental Information Kinesin 1 Drives Autolysosome Tubulation Wanqing Du, Qian Peter Su, Yang Chen, Yueyao Zhu, Dong Jiang, Yueguang Rong, Senyan Zhang, Yixiao Zhang, He Ren, Chuanmao Zhang, Xinquan Wang, Ning Gao, Yanfeng Wang, Lingfei Sun, Yujie Sun, and Li Yu
2 Supplemental Inventory Figure S1. KIF5B localizes to LAMP1-positive organelles. Related to Figure 1A-C. Figure 1A-C showed that KIF5B could rescue the defect of autolysosome reformation in kif5b knock out cells. Figure S1 showed the localization of KIF5B in support of its functions in cells.! Figure S2. WT and T92N KIF5B both localize to purified autolysosomes. Related to Figure 2H, K. Figure 2H, K showed that KIF5B WT but not KIF5B T92N could drive autolysosome tubulation in vitro. Figure S2 showed that both KIF5B WT and KIF5B T92N, but not an irrelevant protein mouse IgG localized to purified autolysosomes in vitro, which supported that tubulation required both localization and motor activity.! Figure S3. Knockdown of kinesin light chain 1 or 2 (KLC1/KLC2) does not affect autolysosome tubulation. Related to Figure 3A, B. Figure 3A, B showed that purified autolysosomes from pip5k1b knockdown cells cannot be tubulated in the in vitro reconstitution system. Figure S3 showed that interfering with other Kif5B adaptors has no effect on autolysosome tubulation, which indicates KIF5B may regulate autolysosome tubulation through PtdIns(4,5) 2. Figure S4. Determination of the lipid binding specificity of KIF5B with a protein lipid overlay assay. Related to Figure 3C, D. Figure 3C, D showed binding of KIF5B to PtdIns(4,5)P 2. Figure S4 showed the lipid binding specificity of KIF5B.! Figure S5. Mapping of the PtdIns(4,5)P 2 -binding domain of KIF5B using a liposome flotation assay. Related to Figure 3C, D. Figure 3C, D showed binding of KIF5B to PtdIns(4,5)P 2. Figure S5 showed detalied domain mapping of PtdIns(4,5)P 2 -KIF5B binding.! Fig. S6. Analysis of CRISPR/Cas knock out KIF5B cell line. Related to Figure 1. The KIF5B knock out cell lines used in Figure 1 was analyzed in Figure S6.! Movie S1.! Supplemental Experimental Procedures. The primer sequence used in cloning, the plasmids cloned in this study and the RNAi sequence used in this study were listed.
3 Figure S1 A B NRK 8h! 8h 0 C +KIF5B T92N! +KIF5B T92N! 8h! 0h +KIF5B WT! +KIF5B T92N! 0h! KIF5B WT! 8h! NRK! +KIF5B WT! 0h! % of KIF5B positive! LAMP1 structures! NRK 0h! KIF5B! KIF5B! LAMP1! Actin! Fig. S1. KIF5B localizes to LAMP1-positive organelles. Related to Figure 1A-C (A) NRK cells and kif5b knockout cells stably expressing WT KIF5B or KIF5B T92N were starved for 0h or 8h, treated with 25 μg/ml digitonin and stained with antibodies against KIF5B and LAMP1. Scale bar, 5 μm. (B) Autolysosomes from A were assessed for the presence of KIF5B puncta and quantified. n> 500 autolysosomes from three independent experiments were quantified. Error bars indicate the s.d. The result that KIF5B could rescue the phenotype of kif5b knock out cells were depicted in Figure 1A-C. (C) The KIF5B protein expression level was tested by Western Blot using anti-kif5b antibody.
4 Figure S2 MTs! Autolysosomes! KIF5B! Merge! MTs! Autolysosomes! KIF5B T92N! Merge! MTs! Autolysosomes! Mouse IgG! Merge! 2μm Fig. S2: WT and T92N KIF5B both localize to purified autolysosomes. Related to Figure 2H, K. Purified autolysosomes were stained by CM-DiI (yellow), incubated with a mixture of nonfluorescent labeled KIF5B and Atto488 labeled KIF5B WT or incubated with Atto488 labeled KIF5B T92N, and transferred into flow chamber channels coated with Hilyte Fluor 647-labeled microtubules (gray). Purified autolysosomes were stained by CM-DiI (yellow), incubated with a mixture of nonfluorescent labeled KIF5B and Atto488 mouse IgG as negative control. The images were collected with a Nikon TIRF microscope. Scale bar, 2μm. Figure 2H, K showed that KIF5B WT but not KIF5B T92N could drive autolysosome tubulation in vitro.
5 Figure S3 A NS! 8h KLC2! RNAi! 8h NS! 8h! KLC1! RNAi! 8h! LAMP1 B % Cells with tubular! autolysosomes C % KLC2 mrna level D! % Cells with tubular! autolysosomes! 80! 80! 60! 60! 40! 40! 20! 20! 0! 0! NS!KLC1! % KLC1 mrna level! E! 120! 80! 80! 40! 40! 0! 0! Fig. S3. Knockdown of kinesin light chain 1 or 2 (KLC1/KLC2) does not affect autolysosome tubulation. Related to Figure 3A, B. (A) NRK cells stably expressing LAMP1-mCherry (Red) were transfected with nonspecific (NS)- or klc1/2-rnai and starved for 8 h. Scale bar, 5 μm. (B)(D) Cells from A were assessed for tubular autolysosomes after 8 h starvation and quantified. n=100 cells from three independent experiments. Error bars indicate the s.d. (C)(E) Cells were transfected with non-specific (NS) RNAi or RNAi against klc1/2. After 60 h of transfection, mrna levels were measured by qpcr. qpcr was performed in triplicate and the data represent the mean ± s.d. This experiment was repeated 3 times. (D) mrna levels of KLC1 and KLC3 in NRK cells were determined by RNAseq. Beta-actin was used as the control. This result supplement Figure 3A, B which showed that lacking pip5k1b interfered with autolysosome tubulation in vitro. Figure S3 showed that interfered with other Kif5B adaptors has no effect on autolysosome tubulation.
6 Figure S4 Triglyceride DAG PA PS PE PC PG Cardiolipin PI PtdIns4P PtdIns(4,5)P 2 PtdIns(3,4,5)P 3 Cholesterol Sphingomyelin Sulfatide Blue Blank Fig. S4. Determination of the lipid binding specificity of KIF5B with a protein lipid overlay assay. Related to Figure 3C, D. The PIP strip was incubated with 2 μg/ ml purified KIF5B in 20 mm Tris-HCl, ph 7.4, 150 mm NaCl, supplemented with 10% (w/v) milk powder for 1 h at room temperature. Binding was detected with an anti- KIF5B antibody and an HRP-conjugated secondary antibody. The binding of KIF5B to PtdIns(4,5)P 2 was carefully analyzed in Figure 3C, D.
7 Figure S5 GST-! ! Protein+Liposome! Protein only! 55! 35! GST-! Cargo domain! Protein+Liposome! Protein only! 70! 0%! His-1-813! His-! Motor domain! Protein+Liposome! Protein only! Protein+Liposome! Protein only! 100! 70! 20%! 25%! 30%! GST! Protein+Liposome! 25! 1 Protein only! 555! 560! 814! 963! Motor domain! Cargo-binding domain! Fig. S5. Mapping of the PtdIns(4,5)P 2 -binding domain of KIF5B using a liposome flotation assay. Related to Figure 3C,D. Main panel: Retention of different domains of KIF5B by 25% PtdIns(4,5)P 2 liposomes in a sucrose gradient. The GST protein serves as a negative control for detecting nonspecific binding. Each fraction of the total sample was visualized by Coomassie staining. When a protein binds to liposomes, it is found in the top fractions. In contrast, if there are no interactions, the protein is localized in the bottom four fractions. Right panel: Schematic representation of sucrose gradient. Bottom panel: Schematic representation of different KIF5B domains used in this experiment. The binding of KIF5B to PtdIns(4,5)P 2 was carefully analyzed in Figure 3C, D.
8 Figure S6 A AATTCTACTTTGTTTTCTATGTAGGGTAAACTTCATGATCCAGAAGGCATGGGGATTATTCC (Wild-type)! KIF5B - /- B AATTCTACTTTGTTTTCTATGTAGGGTAAACTTCATGATC-AGAAGGCATGGGGATTATTCC (Δ1, 3/10)! AATTCTACTTTGTTTTCTATGTAGGGTAAACTTCATGAT-CAGAAGGCATGGGGATTATTCC (Δ1, 7/10)! grna! On-target! Targeted sequence (with PAM)! Genomic Site! Locus! KIF5B! TRUE! GTAAACTTCATGATCCAGA AGG! chr17: ! NM_057202! N/A! Mean Indels (%)+SD! FALSE! GGTGGACTTGAAGATCCAGA GGG! chr1: ! NM_ ! 0! FALSE! GTTAACCTCCATCATCCAGA TAG! chr15: ! NM_ ! 0! FALSE! GGAAAATTTCATTATCTAGA AGG! chr1: ! NM_ ! 0! FALSE! GGAAAATTTCATTATCTAGA AGG! chr2: ! NM_ ! 0! FALSE! GGAAGACTTCATGATAGAGA AAG! chr1: ! NM_012693! 0! FALSE! GGTAAACATCATGATGAAAA AAG! chr4: ! NM_181381! 0! FALSE! GGTATACTTCATGGTGCAAA GGG! chr2: ! NM_ ! 0! Fig. S6. Analysis of CRISPR/Cas knock out KIF5B cell line. Related to Figure 1. (A) DNA sequencing analysis of mutated alleles in the Rat kif5b locus. Partial coding sequences of targeted genes in the genome containing the CRISPR/Cas9 grna KIF5B binding region (underlined) and sequencing analysis of the mutated alleles from a single clone of KIF5B knock out cells were shown. Primers used for the PCR reactions are listed in Supplemental Experimental Procedures, and the red nucleotides represent the PAM sequences that guide the Cas9 for DNA recognition and cleavage. (B) Off-target analysis of 7 potential off-target sites. All on-target and off-target sites were individually amplified from genomic DNA from KIF5B knock out NRK cells. Off-target sites were chosen based on analysis using the CRISPR design tool ( crispr.mit.edu/), and the seven potential off-target sites of the sgrna were selected for T7E1 assay to calculate indels with ImageJ program. The mismatch bases were highlighted in red and the PAM site is highlighted in blue. Primers used to amplify the off-target sites are listed in Supplemental Experimental Procedures. The kif5b knock out cell lines were used in Figure 1.
9 Movie S1 Movie S1. Taxol-stabilized microtubules moving over a lawn of full-length KIF5B. Related to Figure 2B. Full-length KIF5B was introduced into flow chambers, followed by Hylite Fluor 647-labled microtubules. An ATP mixture containing 20 μm ATP was subsequently injected into the chambers, and the images were recorded every 0.5 s for 55 s using a Nikon Ti-E TIRF microscope. Scale bar, 5 μm.
10 Supplemental Experimental Procedures Primer sequence: Name P1 5'KIF5B Motor_NDE1 GGAATTCCATATGGCGGACCCGGCGGAGTGCAAC P2 3'KIF5B Motor_Xho1 CCGCTCGAGTTTTAGTAATGATGCCATCATTTCAGCT P3 5'KIF5B pfast_bamh1 CGCGGATCCATGCACCACCACCACCACCACATGGCGGACCCGGCG GAG P4 3'KIF5B pfast_xba1 CTAGTCTAGATCACGCCTGCTTGCCTCCAC P5 5'KIF5B T92N CATCTGGAAAGAACCACACAATGGAGGGTAAACTT P6 3'KIF5B T92N CATTGTGTGGTTCTTTCCAGATGATGTTTGTCCA P7 5'pGEX- 4T- 1 KIF5B CGCGGATCCAGCGCTGAGGTGGACTCAG P8 3'pGEX- 4T- 1 KIF5B AAGGAAAAAAGCGGCCGCTCACGCCTGCTTGCCTCCAC P9 5'pGEX- 4T- 1 KIF5B cargo CGCGGATCCATGGCATCATTACTAAAAGACCTT P10 3'pGEX- 4T- 1 KIF5B cargo AAGGAAAAAAGCGGCCGCTCACGCCTGCTTGCCTCCAC P11 5'pCAG- RatKif5A_MLUI CGATACGCGTATGGCGGAGACCAATAACGAATG P12 3'pCAG- RatKif5A_NotI CGATGCGGCCGCTTAGCTGGCTGCCGTCTCTTGGTGG P13 5'pCAG- RatKif5C_MLUI CGATACGCGTATGGCGGATCCAGCCGAATGC P14 3'pCAG- RatKif5C_NotI CGATGCGGCCGCTTACTTCTGGTAGTGAGTGGAGTTTG P15 5'pCAG- RatKif1A_MLUI CGATACGCGTATGGCTGGGGCCTCTGTGAAGG P16 3'pCAG- RatKif1A_NotI CGATGCGGCCGCTCAGACCCGCATCTGCGCAG P17 5'pCAG- RatKif5B_MLUI CGATACGCGTATGGCGGACCCGGCGGAGTG P18 3'pCAG- RatKif5B_NotI CGATGCGGCCGCTCACGCCTGCTTGCCTCCACC P19 5'Rat KLC1_qPCR1 GCAGGGTCTTGACAATGTTC P20 3'Rat KLC1_qPCR1 GAGCTTCATCTTTCTCATTTCCT P21 5'Rat KLC2_qPCR1 GGCTCAGTCAATGGAGAGAA P22 3'Rat KLC2_qPCR1 ACTGTGGGACTGTCAACTTT P23 KIF5B- On target F ATGAGCTTGGTTAGTAAGCTGT P24 KIF5B- On target R AGAAGCTAAGAACAGACTTGGC P25 KIF5B- Off target 1F GGGACCAGAGTTTCCTTCCG P26 KIF5B- Off target 1R CCACAAGCTCGGAAGACTCA P27 KIF5B- Off target 2F GGAGTACAACGCGATTCAGC P28 KIF5B- Off target 2R GCCGCAGTCATACACCCTAT P29 KIF5B- Off target 3F TCCTTCGTACTGGAGATGAAACC P30 KIF5B- Off target 3R GCTAATGCTAACAAGTTTTTGACC P31 KIF5B- Off target 5F GTTTTCTTGCCATCCTCAATCCAT P32 KIF5B- Off target 5R AATGCTAGAGTCCAGGTGGCTAA P33 KIF5B- Off target 6F CATGGAAGCCGGTTGTGTTTC P34 KIF5B- Off target 6R CACTAGATGCAGTGGTTTCAATGG P35 KIF5B- Off target 7F TGCCTCTGCTACTTAGGCTTT P36 KIF5B- Off target 7R TTGGCACATTTTATTTCTCTGTCCC plasmid information: Name insert primer His- KIF5B motor Rat KIF5B motor domain (1-560) P1, P2 KIF5B WT pfastbac DUAL Rat KIF5B WT P3, P4 KIF5B T92N pfastbac DUAL Rat KIF5B T92N P5, P6 GST- KIF5B Rat KIF5B P7, P8 GST- KIF5B cargo ( ) Rat KIF5B cargo( ) P9, P10 pcag- KIF5B Rat KIF5B P11, P12 pcag- KIF5A Rat KIF5A P13, P14 pcag- KIF5C Rat KIF5C P15, P16 pcag- KIF1A Rat KIF1A P17, P18 RNAi sequence: PIP5K1B RNAi clathrin heavy chain RNAi Rat KLC2 Rat KLC1 CCGCTGGCTTTCCGCTACT CAGAGCCATGCTGTCTGCTAATATT TGCTATTCATGAGTCAGATCCGAAA GCATGATCCGCAAGTCCTT
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Supplementary Figure 1
Supplementary Figure 1 Product purity of cytosine base editing for the wheat genomic loci tested. Product distributions and indels frequencies at four representative wheat genomic DNA sites in wheat protoplasts
Supplemental Data. Liu et al. (2013). Plant Cell /tpc
Supplemental Figure 1. The GFP Tag Does Not Disturb the Physiological Functions of WDL3. (A) RT-PCR analysis of WDL3 expression in wild-type, WDL3-GFP, and WDL3 (without the GFP tag) transgenic seedlings.
Supplementary Materials for
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Supplementary Figure 1 Endogenous gene tagging to study subcellular localization and chromatin binding. a, b, Schematic of experimental set-up to endogenously tag RNAi factors using the CRISPR Cas9 technology,
Supplementary Information
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Supplementary 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
Supplementary Figure 1. Confirmation of sirna in PC3 and H1299 cells PC3 (a) and H1299 (b) cells were transfected with sirna oligonucleotides
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Supplementary Material
Supplementary Material Supplementary Methods Cell synchronization. For synchronized cell growth, thymidine was added to 30% confluent U2OS cells to a final concentration of 2.5mM. Cells were incubated
Supplementary Information
Supplementary Information Super-resolution imaging of fluorescently labeled, endogenous RNA Polymerase II in living cells with CRISPR/Cas9-mediated gene editing Won-Ki Cho 1, Namrata Jayanth 1, Susan Mullen
SUPPLEMENTARY INFORMATION. Small molecule activation of the TRAIL receptor DR5 in human cancer cells
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At E17.5, the embryos were rinsed in phosphate-buffered saline (PBS) and immersed in
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B. ADM: C. D. Apoptosis: 1.68% 2.99% 1.31% Figure.S1,Li et al. number. invaded cells. HuH7 BxPC-3 DLD-1.
A. - Figure.S1,Li et al. B. : - + - + - + E-cadherin CK19 α-sma vimentin β -actin C. D. Apoptosis: 1.68% 2.99% 1.31% - : - + - + - + Apoptosis: 48.33% 45.32% 44.59% E. invaded cells number 400 300 200
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 Wang et al., http://www.jcb.org/cgi/content/full/jcb.201405026/dc1 T H E J O U R N A L O F C E L L B I O L O G Y Figure S1. Generation and characterization of unc-40 alleles. (A and
S156AT168AY175A (AAA) were purified as GST-fusion proteins and incubated with GSTfused
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 Supplemental Materials Supplemental Figure S1 (a) Phenotype of the wild type and grik1-2 grik2-1 plants after 8 days in darkness.
Supplementary
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MEFs were treated with the indicated concentrations of LLOMe for three hours, washed
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Supplemental Data. Lee et al. Plant Cell. (2010) /tpc Supplemental Figure 1. Protein and Gene Structures of DWA1 and DWA2.
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Supplementary Figure 1 Sox2 localizes in neutrophils of mouse spleen. (a) Microscopy analysis of Sox2 and MPO in wild-type mouse spleen. Nucl, nucleus. (b) Immunohistochemistry staining of Sox2 and MPO
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Supplementary 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
pt7ht vector and over-expressed in E. coli as inclusion bodies. Cells were lysed in 6 M
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Supplemental Information
Supplemental Information ATP-dependent unwinding of U4/U6 snrnas by the Brr2 helicase requires the C-terminus of Prp8 Corina Maeder 1,3, Alan K. Kutach 1,2,3, and Christine Guthrie 1 1 Department of Biochemistry
M X 500 µl. M X 1000 µl
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Supplemental Data. Wu et al. (2). Plant Cell..5/tpc RGLG Hormonal treatment H2O B RGLG µm ABA µm ACC µm GA Time (hours) µm µm MJ µm IA
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DOI: 10.1038/ncb3209 Supplementary Figure 1 IR induces the association of FH with chromatin. a, U2OS cells synchronized by thymidine double block (2 mm) underwent no release (G1 phase) or release for 2
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p Kinesin light chain Kinesin heavy chain Kinesin heavy chain Kinesin light chain + - BCMA
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Supplementary Information stability is regulated by CK2-dependent interaction with R2TP complex Patrick von Morgen 1,2, Kamila Burdova 1, Thomas G. Flower 3, Nicola J. O'Reilly 4, Simon J. Boulton 5, Stephen
Zhang et al., RepID facilitates replication Initiation. Supplemental Information:
Supplemental Information: a b 1 Supplementary Figure 1 (a) DNA sequence of all the oligonucleotides used in this study. Only one strand is shown. The unshaded nucleotide sequences show changes from the
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Khaled_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
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