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Figure S1

siRNA:. Control . Chk1 . TdR(h): 30 1 6 16 24 30 48 . - + - + - + - + - + - + - + . Chk1 . γ H2AX . RPA34 . Cleaved casp3 . β -actin . siRNA:. Control . Chk1 .

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Figure S1

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  1. siRNA: Control Chk1 TdR(h): 30 1 6 16 24 30 48 - + - + - + - + - + - + - + Chk1 γH2AX RPA34 Cleaved casp3 β-actin siRNA: Control Chk1 TdR(h): 30 1 6 24 30 48 - + - + - + - + - + - + Chk1 Chk1 γH2AX γH2AX RPA34 RPA34 Cleaved casp3 Cleaved casp3 β-actin β-actin Chk1 inhibitor (Gö6976) 0.1 mM TdR(h): 30 1 6 16 24 30 10Gy - - - - - - + + + + + + FigureS1 A MRC5 B SW480 C HCT116 Figure S1. gH2AX formation is enhanced in Chk1-depleted or Chk1 inhibited cells during replication stress. Western blot analysis of γH2AX, RPA 34 and cleaved caspase 3 in extracts obtained from MRC5VA (A) or SW480 (B) cells transfected with control or Chk1 siRNAs and treated or not treated with 2 mM thymidine for the indicated times. (C) Western blot analysis of extracts from HCT116 cells treated with the Chk1 inhibitor. Levels of H2AX and RPA34 in extracts of cells exposed to 10 Gy IR are also presented. The levels of Chk1 in the all the cells are presented and -actin levels are presented as loading controls.

  2. Chk1 depleted HCT116 Min post IR - 15 30 60 90 120 Chk1 Control Control Control Chk1 Chk1 siRNA: Control Chk1 Chk1 Control Time(min) after IR (10Gy): - - 30 30 60 60 90 90 120 120 γH2AX Chk1 RPA34 γ-H2AX Merge RPA34 β-actin Figure S2 A Control HCT116 cells Min post IR - 15 30 60 90 120 B γH2AX RPA34 Merge C Figure S2. Induction of γH2AX formation and foci in Chk1 depleted cells exposed to IR. (A) Western blot analysis of H2AX phosphorylation in HCT116 cells treated with control or Chk1 siRNAs for 24 hours before IR. Cells were harvested at the indicated times post IR. Chk1 depletion had little effect on the transient formation of H2AX following IR. (B & C) Representative images of IR-induced γH2AX foci formation at the indicated times in control (B) or Chk1 (C) siRNA transfected HCT116 cells. Nucleus is co stained for RPA34. Formation of gH2AX foci occurs at 15 to 90 minutes post IR exposure and is not affected by Chk1 depletion, while RPA foci are not detected. Thus the nuclear organization of H2AX and RPA34 are distinctly different in cells exposed to IR relative to those exposed to replication stress.

  3. B A Chk1-depleted -TdR Chk1-depleted +TdR Control-depleted -TdR Control-depleted +TdR % TUNEL + cells % cells cleaved casp-3 Time(h) in thymidine Time(h) in thymidine 50 50 45 45 40 40 35 35 30 30 25 25 20 20 15 15 10 10 5 5 0 0 0 10 20 30 40 50 60 0 10 20 30 40 50 60 Figure S3 Figure 3. Induction of apoptosis in Chk1 depleted HCT116 cells following thymidine treatment. Cultures of HCT116 cells transfected with control or Chk1 siRNAs were treated with 2mM thymidine for the indicated times or left untreated as controls. Cells were then harvested and the level of apoptotic cells was determined by flow cytometry measuring the percentage of cells with active caspase-3 (A) or TUNEL+ (B). Results represent the means of three independent experiments + standard deviations.

  4. HCT116 Chk1-depleted cells DNA γH2AX merge TdR (h): I (1-10 foci) II (>10 foci) III (diffused pan nuclear staining) II II 6h II II 6h I I II 6h II II 16h II III 16h 16h I III Figure S4 Figure S4. Representative images of H2AX stained HCT116 cells that were depleted of Chk1 and treated with 2mM thymidine for the indicated times before analysis. H2AX forms distinct foci at early times (6h) that becomes more diffuse at longer exposures (>16h). Cells with pan-nuclear staining (III) become more frequent at these later times.

  5. Figure S5 SW480 C A B Figure 4. Cdc45 suppresses the enhanced induction of H2AX phosphorylation during DNA replication stress in the absence of Chk1. Western blot analysis of γH2AX and RPA34 in extracts obtained from the indicated cells transfected with control, Cdc45, or Chk1 siRNAs or treated with the Chk1 inhibitor and exposed to 2 mM thymidine for 24h. The levels of Chk1 and Cdc45 proteins in the cells also are presented, while -actin levels are presented as loading controls. (A) HCT116 cells treated a second Cdc45 siRNA. (B) HCT116 cells treated with the Chk1 inhibitor. (C) SW480 cells treated with Cdc45 and/or Chk1 siRNAs.

  6. Chk1/Cdc45 Chk1/Cdc45 Cdc45 Cdc45 Chk1 Chk1 control control DNA content DNA content FigureS6 B A Gated R3 region cells siRNA: control Chk1 Cdc45 Chk1/Cdc45 untreated 5.2% 5.6% 10.9% 6.5% γH2AX DNA content 24hTdR 58.3% 14.4% 9.1% 11.3% Figure S6. Cdc45 depletion suppresses the enhanced formation of γH2AX in Chk1 depleted cells treated with thymidine and the fraction of cells in mid S-phase. (A) Scatter plotspresent the flow cytometric analysis of γH2AX levels and DNA content (propidium iodide staining) in HCT116 cells transfected with the indicated siRNAs and cultured in thymidine-free (top) or thymidine containing (bottom) media for 24h. Cell cycle profiles (filed histograms below the corresponding γH2AX assay) for all cells analysed in this experiment. (B) Histograms presenting DNA content of cells staining for increased γH2AX (boxed R3 region in A).

  7. Chk1 Chk1 Chk1 control control control Hours in TdR control Chk1 G2/M S 100% 100% G1 80% 80% 0 SubG1 60% 60% 40% 40% 20% 20% 0% 0% 24 24 100% 100% 100% 100% 100% 80% 80% 80% 80% 80% 60% 60% 60% 60% 60% 40% 40% 40% 40% 40% 20% 20% 20% 20% 20% 0% 0% 0% 0% 0% 100% 100% 100% 80% 80% 80% 60% 60% 60% 40% 40% 40% 20% 20% 20% 0% 0% 0% Release in TdR-free medium (h) Continuous exposure to TdR(h) control Chk1 Chk1 control 8 32 40 48 64 16 24 40 Figure S7 Hours in TdR 0 A Release in TdR-free medium (h) Continuous exposure to TdR(h) 8 16 24 40 32 40 48 64 B Figure S7.Chk1-depleted cells show a reduced ability to re-enter the cell cycle after release from thymidine treatment. (A) Flow cytometric analysis of DNA content of HCT116 cells transfected with control or Chk1 siRNAs and treated or not treated with 2mM thymidine (TdR) for 24h (top panels). Cells were then either washed and transferred to fresh medium without thymidine (right panels) or cultured in the continued presence of thymidine (left panels) for the indicated times. (B) Bar graphs presenting the cell cycle distributions of HCT116 cells treated as described in panel A. The results are the means of three independent experiments + standard deviations.

  8. Chk1 siRNA Control siRNA 2.7% 1.4% untreated 4.6% 31.2% 40hTdR 24hTdR, 24h Release 3.2% 24.1% Active casp-3 DNA content Figure S8 Figure S8. Activation of caspase 3 in Chk1-depleted cells released from thymidine. Scatter plots present levels of cleaved caspase 3 and DNA content (propidium iodide staining) determined by flow cytometry in control or Chk1 siRNA transfected HCT116 cells, continuously exposed to thymidine (TdR) for 40h or exposed for 24h and then released into thymidine-free medium for 24h. Untreated cells were used as negative controls. The percentages of cells with activated caspase-3 (boxed R4 region) are presented.

  9. 6h, R40h 46h 6h 0h Figure S9 B Control siRNA Control siRNA A 6h, R40h 16h, R30h 46h 30h 6h 16h 0h 0h Chk1 siRNA Chk1 siRNA 16h, R30h 30h 16h 0h Figure S9. Chk1-depleted cells recover from a short (6h) exposure to thymidine but only show partial recovery from a 16h arrest. HCT116 cells transfected with control or Chk1 siRNAs were treated with 2mM thymidine (TdR) for 6h (A) or 16h (B) and then released into thymidine-free medium for the indicated times or were left to grow continuously in the inhibitor before being harvested and analysed for DNA content. Control cells thymidine-arrested were able to recycle and fully recover from replication stress after release in a thymidine-free medium. Chk1 depleted cells exposed to thymidine for 6h recovered completely after release but those exposed for 16h showed only a partial recovery. Cells transfected with the control siRNA recovered completely after 6 or 16h exposure.

  10. G1: 6.2 S: 28.9 G2: 53.44 G1: 34.9 S: 24.2 G2: 27.65 G1: 14.4 S: 64.1 G2: 10.73 88.6% 86.7% 89.3% G1: 5.7 S: 27.1 G2: 4.56 G1: 10.9 S: 22.9 G2: 2.48 37.4% 36.2% G1: 10.2 S: 21.4 G2: 11.54 43.2% Figure S10 A SW480 24h thymidine Control siRNA Chk1 siRNA h post BrdU pulse 0 4 24 0 4 20 Total cells B SW480 24h HU Control siRNA Chk1 siRNA h post BrdU pulse 0 4 24 0 4 20 Figure S10. Chk1 depleted SW480 cells released from thymidine treatment show suppressed re-entry into S-phase. SW480 cells transfected with control or Chk1 siRNAs were exposed to 2mM thymidine (A) or 2mM HU (B) for 24h. The cells were then washed with inhibitor-free medium and transferred to medium containing 10 mM BrdU. After 1h these cells were washed with BrdU-free medium and harvested for FACS analysis at the indicated times. Representative scatter plots (upper panels) show BrdU incorporation and DNA content (PI staining) in cells transfected with control or Chk1 siRNAs treated with thymidine (A) or HU (B). Cells incorporating BrdU have been gated (as indicated) and the percentages with G1, S, or G2 DNA content are presented together with the percentages of all cells incorporating BrdU. Cells with a G1 DNA content incorporating BrdU at early times after the pulse most likely represent cells at the G1/S border. Lower panels present DNA content (PI staining) of all the cells in the cultures (gated and ungated).

  11. H2AX+ Control Chk1 siRNA: H2AX+ with SubG1 TdR(24h): HU(24h) IR(10Gy) Release(24h) - - + + - - - + + - - - - - - + + - - - + + + - - - - - - - - - - - - - + - + - - + - + Chk1 γH2AX RPA34 pSer1981 ATM pThr68 Chk2 β-actin 100 100 90 90 1-10 foci 80 80 >10 foci 70 70 60 TdR (h): - 24 24 60 pan nuclear % cells % cells 50 50 Release (h): - - 24 40 40 30 30 20 20 Chk1 siRNA 10 10 0 0 TdR(h): 0 0 24 24 24 24 24 24 HU(h): 0 0 24 24 24 24 24 24 Release. (h): 0 0 0 0 24 24 40 40 Release (h): 0 0 0 0 24 24 40 40 γ-H2AX γ-H2AX γ-H2AX siRNA: siRNA: Control Control Control Control Control Control Chk1 Chk1 Control Control Chk1 Chk1 Chk1 Chk1 Chk1 Chk1 Control siRNA γ-H2AX γ-H2AX γ-H2AX C 60 % cells with H2AX foci 50 40 30 20 10 Chk1 Chk1 Chk1 Control Control Control 0 siRNA: untreated TdR24h R24h TdR24h Figure S11 SW480 A B D

  12. Figure S11. Persistence of gH2AX and other DNA damage response proteins in Chk1-depleted SW480 cells following release from thymidine arrest. (A) Western blot analysis of γH2AX, RPA34, phospho-ATM (pSer1981), and phospho-Chk2 (pThr68) in extracts obtained from SW480 cells transfected with control or Chk1 siRNAs and treated or not treated with 2 mM thymidine (TdR) or 2mM HU for 24h before release into thymidine-free medium for the indicated times. In addition extracts from SW480 cells treated with 10 Gy IR and then cultured for 1h before harvest are included. The levels of Chk1 protein in all the cells also are presented while -actin levels are presented as loading controls. (B) Representative immunofluorescence images of γH2AX nuclear distribution in siRNA-transfected SW480 cells treated with thymidine for 24h or treated and released from thymidine for 24h. (C) Percentages ofSW480 cells treated with control or Chk1 siRNAs presenting low (1-10 foci/cell) or high (>10 foci/cell) levels of γH2AX foci, or showing pan-nuclear staining for γH2AX during and after exposure to thymidine for the indicated times. Results presented are the means of three independent experiments + standard deviations. (D)) Percentages (%) of γH2AX positive SW480 cells and gH2AX+ SW480 cells with a subG1 DNA content following treatment with 2mM thymidine or 2mM HU. Cells were treated with the indicated siRNAs before treatment with the replication inhibitor for 24h before release for 24 or 40h. Cells harvested were analysed for DNA and H2AX content by flow cytometry. Results represent the means of three independent experiments + standard deviations.

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