Supplementary Figure 1
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1 Supplementary Figure 1 a γ-h2ax MDC1 RNF8 FK2 BRCA1 U2OS Cells sgrna-1 ** 60 sgrna % positive Cells (>5 foci per cell) b ** 80 sgrna sgrna γ-h2ax MDC1 γ-h2ax RNF8 FK2 MDC1 BRCA1 RNF8 FK2 BRCA1 HeLa Cells sgrna-1 sgrna sgrna ** ** 60 sgrna γ-h2ax MDC1 RNF8 FK2 BRCA1 80 % positive Cells (>5 foci per cell)
2 Supplementary Figure 1. regulates -BRCA1 foci formation in response to cisplatin. (a-b) Control or knockout U2OS (a) or HeLa (b) cells were treated with cisplatin and foci formation of the indicated factors were detected by immunofluorescence with indicated antibodies. Representative images are shown in the upper panels. Scale bar, 10 μm. Quantification of the percentage of cells displaying foci formation is shown in the lower left panels. expression in these cells were examined by western blot and is shown in the lower right panel. Error bars represent SEM from three independent experiments. **P < >200 cells were counted per experiment. Statistical analyses were performed with the ANOVA.
3 Supplementary Figure 2 a γ-h2ax BRCA1 CCDC98 % positive Cells (>5 foci per cell) sgrna sgrna Olaparib c 80 γ-h2ax MDC1 BRCA1 sgrna 20 γ-h2ax b sgrna: sgrna 10uM d sgrna sgrna (hrs) 1uM sgrna H3 I-SceI BRCA1 MERIT 40 Ƴ-H2AX CCDC98 CCDC98 BRCA1 Chromatin BRCA1 10uM ** ** 40 IR: 1uM sgrna 60 0 IR treated (2Gy) sgrna ** Merge
4 Supplementary Figure 2. regulates BRCA1-A complex recruitment to DSBs. (a) Control or knockout EFO-27 cells were treated with IR (2Gy) and foci formation of the indicated factors were detected by immunofluorescence with indicated antibodies. Representative images are shown in the left panels. Quantification of the percentage of cells displaying foci formation is shown in the right panels. Scale bar, 10 μm. Error bars represent SEM from three independent experiments. **P < >200 cells were counted per experiment. Statistical analyses were performed with the ANOVA. (b) Control or knockout EFO-27 cells were treated with IR (2Gy). Whole cell lysates and chromatin fractions were prepared and blotted with the indicated antibodies. (c) Control or knockout EFO-27 cells were treated with olaparib at indicated concentrations for 6 hr. Foci formation of the indicated factors was detected by immunofluorescence with indicated antibodies. Scale bar, 10 μm. (d) Colocalization of BRCA1 with γ-h2ax at DSB site created by I-SceI was examined in control or knockout cells. Scale bar, 10 μm. expression in these cells was examined by western blot and is shown in the panel to the left.
5 Supplementary Figure 3 a (sec) GFP- GFP- b Relative fluorescence intensity GFP- GFP (s)
6 Supplementary Figure 3. Recruitment kinetics of and. (a-b) Cells expressing GFP- or GFP- were subjected to laser microirradiation. Laser stripes were examined at the indicated time points. Scale bar, 10 μm. The intensity of each laser stripe at each time point was determined by averaging values from 10 cells and is graphed in (b).
7 Supplementarg Figure 4 a sgrna: HA- - - WT CA b sgrna: HA- - - WT CA d IP: IgG sgrna: anti-ub HeLa c γ-h2ax WT CA sgrna IP: Lysate % positive Cells (>5 foci per cell) anti-ub U2OS 120 sgrna+ WT γ-h2ax sgrna+ CA ** 2 1 IP: Lysate BRCA1 CCDC98 MERIT 40 anti-ub BRCA1 CCDC98 MERIT IP Input
8 Supplementary Figure 4. Deubiquitination of by affects foci formation but not BRCA1-A complex stabilization. (a-b) HeLa (a) or U2OS (b) cells stably expressing control, sgrna, or sgrna with the indicated constructs, were lysed under denaturing conditions and was immunoprecipitated. Blots were probed with the indicated antibodies. (c) EFO-27 cells stably expressing sgrna with the indicated constructs were treated with cisplatin and foci formation of the indicated factors were detected by immunofluorescence with indicated antibodies. Representative images are shown in the left panel. Scale bar, 10 μm. Quantification of the percentage of cells displaying foci formation is shown in the right panel. Error bars represent SEM from three independent experiments. **P < >200 cells were counted per experiment. Statistical analyses were performed with the Student s t-test. (d) Cell lysates from control () or knockout EFO-27 cells were subjected to immunoprecipitation with control IgG or antibodies. The western blots were then blotted with the indicated antibodies.
9 Supplementary Figure 5 a 0Gy 2Gy d 0Gy 2Gy sgrna sgrna sgrna sgrna sgrna + WT sgrna sgrna + CA sgrna + sgrna sgrna: sgrna : b c Surivival Fraction (%) 10 sgrna sgrna sgrna+ WT Surivival Fraction (%) 10 sgrna sgrna sgrna+ WT 1 IR: sgrna+ CA HeLa (Gy) 1 IR: sgrna+ CA U2OS (Gy)
10 Supplementary Figure 5. regulates G2/M checkpoint and radiosensitivity. (a) The representative data for Fig 3a. Scale bar, 0 μm. (b-c) HeLa (b) or U2OS (c) cells stably expressing control, sgrna, or sgrna with the indicated constructs were generated. Sensitivity of these cells to IR was assessed using colony formation assay. Error bars represent SEM from three independent experiments. (d) The representative data for Fig 3d. Scale bar, 0 μm. and expression in these cells were examined by western blot and is shown in the lower panel.
11 Supplementary Figure 6 sgrna sgrna a Homologous Recombination (Normalize to 1) HA- - - WT CA sgrna: HA- - - WT CA sgrna: sgrna+wt sgrna+ca b Homologous Recombination (Normalize to 1) Spautin-1: sgrna: Spautin-1: n.s. sgrna: - sgrna Spautin-1 sgrna+spautin-1
12 Supplementary Figure 6. regulates HR. (a) HR-mediated DSB repair capacity of control, knockout, and knockout cells stably expressing the indicated constructs were assessed using a reporter system. (b) HR-mediated DSB repair capacity of control and knockout cells treated with or without Spautin-1 was assessed using a reporter system. Error bars represent SEM from three independent experiments. *P < Statistical analyses were performed with the ANOVA.
13 Supplementary Figure 7 a sgrna: Olaparib: anti-ub b HA (um) WT 5KR-1 (K20/31/75/90/112R) 2 1 5KR-2 (K5146/232/245/374/382R) 5KR-3 (K544/567/587/607/635R) IP: Lysate c HA- sgrna: anti-ub HA WT 2KR (K20/31R) 3KR (K75/90/112R) d HA- WT 2 1 K75R IP:FLAG Lysate K90R e GST--WT GST--3KR His-K48 Ub chain His-K63 Ub chain K112R anti-his 3KR (K75/90/112R) GST GST His-pulldown Input sgrna: sgrna: anti-ub 2 1 IP:FLAG anti-ub 2 1 IP:FLAG HA Lysate HA Lysate
14 Supplementary Figure 7. Identification of the deubiquitination sites targeted by. (a) Control and knockout cells treated with olaparib at different concentrations were lysed under denaturing conditions and was immunoprecipitated. Blots were probed with the indicated antibodies. (b-d) Control and knockout cells were transfected with indicated constructs. Cells were lysed under denaturing conditions and HA- was immunoprecipitated. Blots were probed with the indicated antibodies. (e) GST- WT and 3KR mutant proteins were subjected to pull down assay by incubating with indicated His-Ub chains conjugated with Ni-NTA beads. After washing, proteins bound on beads were subjected to western blot with indicated antibodies.
15 Supplementary Figure 8 cisplatin p-sq/tq e a % positive Cells (>5 foci per cell) HeLa Ku55933 phosphatase IP: b cisplatin p-sq/tq U2OS Ku55933 phosphatase sgrna sgrna sgrna+ WT sgrna+ T196A 0 γ-h2ax CCDC98 HA- - - WT T196A BRCA1 sgrna: IP: c HA- cisplatin p-t196 HA f Homologous Recombination (Normalize to 1) WT T196A sgrna: d HA- - - WT TA cisplatin IP:HA anti-ub 2 1 IP: Lysate sgrna: HA- - - WT T196A g sgrna sgrna sgrna+wt sgrna+t196a MDC1 shrna h i MDC1 Ƴ-H2AX I-SceI Merge 2 Surivival Fraction (%) 10 1 Olaparib: sgrna sgrna sgrna+wt sgrna+ T196A (um) shrna MDC1 shrna
16 Supplementary Figure 8. Phosphorylation of regulates DDR. (a-b) HeLa (a) or U2OS (b) cells were pretreated with DMSO or 25μM Ku55933 for 2 hrs following which they were left untreated or treated with cisplatin. After an additional 1 hr, was immunoprecipitated, left untreated or treated with phosphatase, and immunoblotted with phospho-sq/tq (psq/tq) antibody. (c) HEK293T cells transfected with HA- WT or T196A mutant were left untreated or treated with cisplatin. HA- was immunoprecipitated and immunoblotted with T196 phospho-specific antibody. (d) Control and knockout cells were transfected with the indicated constructs. Cells were lysed under denaturing conditions and was immunoprecipitated. Blots were probed with the indicated antibodies. (e) EFO- 27 cells stably expressing, sgrna or sgrna with the indicated constructs were treated with cisplatin and foci formation of the indicated factors were detected by immunofluorescence with the indicated antibodies. Error bars represent SEM from three independent experiments. >200 cells were counted per experiment. (f-g) Control, knockout, and knockout cells stably expressing the indicated constructs were subjected to DR-GFP based HR assay. Error bars represent SEM from three independent experiments. **P < 0.01, *P<0.05. Statistical analyses were performed with the ANOVA. expression is shown in the right panel. The representative FACS data is shown in (g). (h) Control, knockout, and knockout cells stably expressing the indicated constructs were subjected to colony formation assay to assess the sensitivity of cells to olaparib. Error bars represent SEM from three independent experiments. (i) Co-localization of with γ-h2ax at DSB site created by I-SceI was examined in control or MDC1 knockdown cells. Scale bar, 5 μm. MDC1 expression in these cells was examined by western blot and is shown in the right upper panel.
17 Supplementary Figure 9 a log2 copy number units log2 copy number units CCDC98 log2 copy number units BRCA1 Normal (561 cases) Ovarian carcinoma (607 cases) Normal (561 cases) Ovarian carcinoma (607 cases) Normal (561 cases) Ovarian carcinoma (607 cases) log2 copy number units MERIT40 log2 copy number units BRCC45 b Normal (561 cases) Ovarian carcinoma (607 cases) Normal (561 cases) Ovarian carcinoma (607 cases)
18 Supplementary Figure 9. The expression of in ovarian cancer. (a) The expression of BRCA1-A complex in normal and ovarian carcinoma (Oncomine data). (b) Gene copy-number and mutation data of and BRCA1-A complex in ovarian cancer (TCGA data).
19 Supplementary Figure 10 a HOSE EFO-27 SKOV-3 FU-OV-1 OVCAR-3 A2780 b HOSE EFO-27 SKOV-3 FU-OV-1 OVCAR-3 A PTEN anti-ub 1 c Survival Fraction (%) 10 shrna USP10 shrna shrna shrna+spautin-1 USP10 shrna+spautin-1 shrna+spautin-1 1 Olaparib: (um) d Absorbance (590nm) (Normalize day 1 to 1) ctrl sgrna sgrna-1 sgrna (Days) e Cell Number sgrna sgrna-1 sgrna-2 G1:43.9 S: G2/M: 8.91 G1:44.12 S: G2/M: 9.69 G1:42.9 S: G2/M: Channels (PI-A)
20 Supplementary Figure 10. regulates chemoresistance in ovarian cancer. (a) The human ovarian epithelial cell line (HOSE) and ovarian carcinoma cell lines were lysed under denaturing conditions and was immunoprecipitated. Blots were probed with the indicated antibodies. (b) Expression of PTEN in human ovarian epithelial cell line and ovarian carcinoma cell lines. (c) Control, knockdown, and USP10 knockdown cells were subjected to colony formation assay to assess the sensitivity of cells to olaparib or olaparib with spautin-1. (d) Growth curves of EFO-27 cells stably expressed ctrl or sgrnas were measured by MTS assay. (c-d) Error bars represent SEM from three independent experiments. (e) Cell cycle profile of EFO-27 cell stably expressed ctrl or sgrnas.
21 Supplementary Figure 11 Fig 1b sgrna -1-2 Fig 2a IP IgG Input IgG Fig 2b Fig 2c sgrna: IP: IgG IgG sgrna: HA- - - WT CA IP: Ub blot Fig 2d sgrna: Spautin-1: Fig 2e IP: - WT CA HA- Ub blot His-Ub blot GST- Fig 2f His-Ub: - K48 K63 K63R 13sgRNA: IP: His-Ub blot His-Ub Input
22 Supplementary Figure 11. Original scan of the blots presented in the main text. Related to Figure 1 and 2.
23 Supplementary Figure 12 Fig 4a sgrna: Cisplatin: IP: Fig 4b sgrna: Ub chain K48 K63 Cisplatin: Ub Blot His-Ub pulldown Input Fig 4d HA- WT 3KR Fig 4f sgrna: sgrna: Ub chain K48 K63 HA-: WT WT 3KR WT 3KR IP:HA- HA- Input His-Ub pulldown Ub Blot HA- Fig 5a cisplatin Ku55933 phosphatase Fig 5b HA- WT T196A T380A T385A cisplatin p-sq/tq p-sq/tq IP: IP:HA-
24 Supplementary Figure 12. Original scan of the blots presented in the main text. Related to Figure 4 and 5.
25 Supplementary Figure 13 Fig 5c ATM+/+ ATM-/- - IR Cisplatin - IR Cisplatin Fig 5d WT T196A HA Fig 5f cisplatin: HA-MDC Input ATM IP:HA-MDC1 p-sq/tq Fig 5h HA-: WT T196A Cisplatin: HA- HA- Input IP: Fig 5g MDC1-FHA GST GST-pulldown Fig 5i Conjugated Peptides Fig 6f T196 p-t196 Input HA-MDC1: - WT D1 D2 D3 D4 D5 D6 D7 D8 GST HA- IP:HA-MDC1 Fig 6b Incubated with GST-MDC1 -FHA HOSE EFO-27 SKOV-3 FU-OV-1 OVCAR-3 A2780 Input
26 Supplementary Figure 13. Original scan of the blots presented in the main text. Related to Figure 5 and 6.
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Supplementary Figure 1. A. Bar graph representing the expression levels of the 19 indicated genes in the microarrays analyses comparing human lung
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doi: 10.1038/nature05732 SUPPLEMENTARY INFORMATION Supplemental Data Supplement Figure Legends Figure S1. RIG-I 2CARD undergo robust ubiquitination a, (top) At 48 h posttransfection with a GST, GST-RIG-I-2CARD
T H E J O U R N A L O F C E L L B I O L O G Y
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Supplementary Figure 1. mir124 does not change neuron morphology and synaptic
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APC/C Cdh1 controls CtIP stability during the cell cycle and in response to DNA damage
Article APC/C Cdh1 controls stability during the cell cycle and in response to DNA damage Lorenzo Lafranchi 1,, Harmen R de Boer 2,, Elisabeth GE de Vries 2, Shao-En Ong 3, Alessandro A Sartori 1,* & Marcel
Supplementary Materials
Supplementary Materials Figure S1. MTT Cell viability assay. To measure the cytotoxic potential of the oxidative treatment, the MTT [3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyl tetrazolium bromide] assay
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Shao-Ming Shen et al Role of I in MT of cancers MO reports xpanded View igures igure V1. nalysis of the expression of I isoforms in cancer cells and their interaction with PTN. RT PR detection of Ish and
hexahistidine tagged GRP78 devoid of the KDEL motif (GRP78-His) on SDS-PAGE. This
SUPPLEMENTAL FIGURE LEGEND Fig. S1. Generation and characterization of. (A) Coomassie staining of soluble hexahistidine tagged GRP78 devoid of the KDEL motif (GRP78-His) on SDS-PAGE. This protein was expressed
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MO reports PR3 dephosphorylates TZ Xian-o Lv et al xpanded View igures igure V1. PR3 dephosphorylates and inactivates YP/TZ., Overexpression of tight junction proteins Pals1 () or LIN7 () has no effect
The clathrin adaptor Numb regulates intestinal cholesterol. absorption through dynamic interaction with NPC1L1
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HCC1937 is the HCC1937-pcDNA3 cell line, which was derived from a breast cancer with a mutation
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HA-Sin1-N (aa 1-137), HA-Sin1-CRIM (aa ) and HA-Sin1-RBD (aa ) were constructed by
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a b G75 G60 Sw-2 Sw-1 Supplementary Figure 1. Structure predictions by I-TASSER Server.
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Control GST GST-RAP. α2-mg. 170 kda. b-actin. 42 kda LRP-1
% of max Supplementary Figure 1 Control GST GST-RP 17 kda α2-mg 42 kda b-actin Gate: CD11c+ (DCs) Gate: F4/8+ (Mfs) IgG Cd11cCre + Lrp1 fl/fl LRP-1 Supplementary figure 1. () MDCs were pretreated with
Supplementary Materials for
www.sciencesignaling.org/cgi/content/full/9/439/ra78/dc1 Supplementary Materials for Small heterodimer partner mediates liver X receptor (LXR) dependent suppression of inflammatory signaling by promoting
IP: anti-gfp VPS29-GFP. IP: anti-vps26. IP: anti-gfp - + +
FAM21 Strump. WASH1 IP: anti- 1 2 3 4 5 6 FAM21 Strump. FKBP IP: anti-gfp VPS29- GFP GFP-FAM21 tail H H/P P H H/P P c FAM21 FKBP Strump. VPS29-GFP IP: anti-gfp 1 2 3 FKBP VPS VPS VPS VPS29 1 = VPS29-GFP
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SUPPLEMENTARY INFORMATION doi:10.1038/nature11429 S1a 6 7 8 9 Nlrc4 allele S1b Nlrc4 +/+ Nlrc4 +/F Nlrc4 F/F 9 Targeting construct 422 bp 273 bp FRT-neo-gb-PGK-FRT 3x.STOP S1c Nlrc4 +/+ Nlrc4 F/F casp1
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SUPPLEMENTARY FIGURES Figure S1. Clinical significance of ZNF322A overexpression in Caucasian lung cancer patients. (A) Representative immunohistochemistry images of ZNF322A protein expression in tissue
Supplementary Figure 1. Confocal immunofluorescence showing mitochondrial translocation of Drp1. Cardiomyocytes treated with H 2 O 2 were prestained
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Supplementary Figure S1: Defective heterochromatin repair in HGPS progeroid cells
Supplementary Figure S1: Defective heterochromatin repair in HGPS progeroid cells Immunofluorescence staining of H3K9me3 and 53BP1 in PH and HGADFN003 (HG003) cells at 24 h after γ-irradiation. Scale bar,
Tel: ; Fax: ;
Tel.: +98 216 696 9291; Fax: +98 216 696 9291; E-mail: mrasadeghi@pasteur.ac.ir Tel: +98 916 113 7679; Fax: +98 613 333 6380; E-mail: abakhshi_e@ajums.ac.ir A Soluble Chromatin-bound MOI 0 1 5 0 1 5 HDAC2
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DOI: 1.138/ncb3445 In the format provided by the authors and unedited. a b HIF1A 14 12 Pearson R =.5419 P
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doi: 10.1038/nature06994 A phosphatase cascade by which rewarding stimuli control nucleosomal response A. Stipanovich*, E. Valjent*, M. Matamales*, A. Nishi, J.H. Ahn, M. Maroteaux, J. Bertran-Gonzalez,
Legends for Supplemental Figures.
1 Legends for Supplemental Figures. Supplemental Figure 1. Proteasome Inhibition does not abrogate the expression of FA core complex proteins or formation of the FA core complex. (Full-length blots are
Incorporation of photo-caged lysine (pc-lys) at K273 of human LCK allows specific control of the enzyme activity.
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F-actin VWF Vinculin. F-actin. Vinculin VWF
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SUPPLEMENTARY FIGURES
SUPPLEMENTARY FIGURES Supplementary Figure S1: Fibroblast-induced elongation of cancer cells requires direct contact with living fibroblasts. A. Representative images of HT29-GFP cultured in the presence
Supplementary Figure S1
Supplementary Figure S1 Supplementary Figure S1. PARP localization patterns using GFP-PARP and PARP-specific antibody libraries GFP-PARP localization in non-fixed (A) and formaldehyde fixed (B) GFP-PARPx
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Supplementary Table 1. Cell sphingolipids and S1P bound to endogenous TRAF2. Sphingolipid Cell pmol/mg TRAF2 immunoprecipitate pmol/mg Sphingomyelin 4200 ± 250 Not detected Monohexosylceramide 311 ± 18
A. Generation and characterization of Ras-expressing autophagycompetent
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Supplementary Figure 1
Supplementary Figure 1 Dox Cis Cam Pac 0 15 1 15 1 15 1 15 1 15 µmole/l Ub p53 Cytotoxic anticancer agents increase p53 levels but do not generally promote the accumulation of ubiquitinated. Western blots
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SUPPLEMENTARY FIGURES AND TABLE Supplementary Figure S1: Characterization of IRE1α mutants. A. U87-LUC cells were transduced with the lentiviral vector containing the GFP sequence (U87-LUC Tet-ON GFP).
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Fig. S1. Subcellular localization of overexpressed LPP3wt-GFP in COS-7 and HeLa cells. Cos7 (top) and HeLa (bottom) cells expressing for 24 h human
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supplementary information
DOI: 1.138/ncb1 Control Atg7 / NAC 1 1 1 1 (mm) Control Atg7 / NAC 1 1 1 1 (mm) Lamin B Gstm1 Figure S1 Neither the translocation of into the nucleus nor the induction of antioxidant proteins in autophagydeficient
Figure 6: TERT regulates MYC half-life and ubiquitination.
TERT or IgG as indicated. For the western blots, representative images of n= independent experiments are shown. Student s t-test was used, and * indicates p
Tbk1-TKO! DN cells (%)! 15! 10!
a! T Cells! TKO! B Cells! TKO! b! CD4! 8.9 85.2 3.4 2.88 CD8! Tbk1-TKO! 1.1 84.8 2.51 2.54 c! DN cells (%)! 4 3 2 1 DP cells (%)! 9 8 7 6 CD4 + SP cells (%)! 5 4 3 2 1 5 TKO! TKO! TKO! TKO! 15 1 5 CD8
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DOI: 1.138/ncb3355 a S1A8 + cells/ total.1.8.6.4.2 b S1A8/?-Actin c % T-cell proliferation 3 25 2 15 1 5 T cells Supplementary Figure 1 Inter-tumoral heterogeneity of MDSC accumulation in mammary tumor