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cmi12028-sup-0001-fS1.tif5697K

Fig. S1. Cell cycle effect of CDT on p53-positive cells.

A. HT1080 cells were exposed to EcCDTWT LD50 for 72 h. Cells were stained with propidium iodid and cell-cycle distribution analysis was performed by flow cytometry. Peaks respectively correspond to cells in G1 (left peak) or in G2 (right peak).

B. HeLa cells were exposed to EcCDTWT LD50 for 24 h and stained with 53BP1 and cyclin A antibodies. Cells positive for cyclin A (white bars), positive for 53BP1 foci formation and cyclin A (light grey bars), or positive for 53BP1 foci formation and cyclin A negative (dark grey bars) were quantified. Cells were scored positive for foci formation when > 5 foci/nuclei were detected. n = 3; results obtained from independent experiments. Error bars, SD; *P < 0.05.

cmi12028-sup-0002-fS2.tif9912K

Fig. S2. Evolution of PCNA and cyclin B1 staining during cell cycle.

A. PCNA staining progresses during S-phase. Example of PCNA pan-nuclear staining (left cell), early S-phase specific, and PCNA foci staining (right cell), late S-phase specific.

B. HeLa cells were synchronized by double thymidine bloc as described in Experimental procedures. Cell were released in fresh medium for the indicated times and stained with an anti-PCNA antibody. Proportion of cells with a pan-nuclear staining (early S, dark grey) or foci PCNA staining (late S, lighter grey) was counted. One representative experiment is shown.

C. Cyclin B1 staining during the cell cycle. Example of various cyclin B1 staining (green): G1 cells are cyclin B1 negative; S-phase cells have a light, cytoplasmic cyclin B1 staining; G2 cells present a strong, cytoplasmic cyclin B1 staining; cells in mitosis show a strong pan-cellular cyclin B1 staining with condensed chromosomes.

D. HeLa cells were synchronized by double thymidine bloc as described in Experimental procedures. Cells were released in fresh medium for the indicated times and stained with an anti-cyclin B1 antibody. Scale bar = 20 μm. According to the cyclin B1 staining, the proportion of cells in S-phase (grey), G2-phase (white) or mitosis (dark grey) has been assessed.

cmi12028-sup-0003-fS3.tif21449K

Fig. S3. RAD51 do not relocalize to direct DSB induced by high concentrations of CDT.

A. HeLa cells were exposed to EcCDTWT for 24 h and DSBs were detected by γH2AX (red) and RAD51 (green) immunostaining. Scale bar = 20 μm.

B. Quantification of HeLa cells positive for γH2AX and RAD51 foci formation after EcCDTWT exposure for 3 h or 6 h and for the following concentrations: LD50 (50 pg ml−1), LD75 (2.4 ng ml−1), LD95 (25 ng ml−1). Results obtained from three independent experiments. Error bars, SD; *P < 0.05, **P < 0.01.

cmi12028-sup-0004-fS4.tif5065K

Fig. S4. Camptothecin induces replication-associated DSB similar to CDT. HeLa cells were exposed to 0.1 μM camptothecin for the indicated times and stained with 53BP1 and PCNA antibodies.

A. HeLa cells positive for PCNA pan-staining (early S-phase cells, dark grey bars) or PCNA foci staining (late S-phase cells, light grey bars) were quantified. Cells were scored positive for PCNA foci when distinguishable foci could be observed and positive for PCNA pan-staining when PCNA stained uniformly the nucleus. n = 3; results obtained from independent experiments. Error bars, SD.

B. Quantification of HeLa cells positive for 53BP1 foci formation. Cells were scored positive for 53BP1 foci formation when > 5 foci/nuclei were detected in cells positive for PCNA foci staining (light grey bars), in cells positive for PCNA pan-nuclear staining (dark grey bars) or in cells negative for PCNA staining (white bars). n = 3; results obtained from independent experiments. Error bars, SD.

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