Fig. 6

(A) HeLa cells treated with indicated doses of MMC were harvested at 9 hrs. FANCI IPs were analyzed by western blot.

(B) HeLa cells treated with indicated doses of MMC were harvested at 24 hrs and analyzed by western blot.

(C) Model derived from the results in panels A-B: FANCI switches from FANCD2 to PIDD1 with increasing ICL levels.

(D-G) HeLa cells synchronized as in Table S1B were transfected with indicated siRNAs, treated with or without MMC (1 ?M) and harvested 9 hrs post-MMC. FANCI IPs were analyzed by western blot.

(D? ? G?) As above but harvested 24 hrs post-MMC and whole-cell lysates analyzed by western blot. cl.C3, cleaved caspase-3.

(H) HeLa cells expressing the indicated shRNAs were transfected with indicated siRNAs, treated with or without MMC (0.1 ?M) and stained with alamarBlue 72 hrs post-treatment. Data are means +/? SD of 3 independent experiments. n.s: non-significant, ***p < 0.001, two-tailed Student?s t-test.

(I-J) HeLa cells synchronized as in Table S1B were transfected with indicated siRNAs and treated with MMC (1 ?M) (I) or Gö6976 and MMC (1 ?M each) (J) and harvested at indicated time points post-MMC. FANCI IPs were analyzed by western blot.

(K) HeLa cells synchronized as in Table S1B were transfected with indicated siRNAs, treated with or without Gö6976 and MMC (1 ?M each) and harvested 9 hrs post-MMC. FANCI IPs were analyzed by western blot.

(L) HeLa cells transfected with indicated siRNAs were treated with or without Gö6976 (1 ?M) and MMC (0.1 ?M). Alamarblue was added 72 hrs post-treatment. Data are means +/? SEM of 3 independent experiments. **p < 0.01 ***p < 0.001, two-tailed Student?s t-test.

(M-N) Schematic diagrams summarizing the results in panels D-I (M) and J-L (N).