Fig 2: Effect of miR146a on FANCD2 monoubiquitination and the FA pathway(A and B) HeLa cells were transfected with miR146a in the absence or presence of anti-miR146a (A) or miR146a-insensitive FANCM cDNA (B). After a 48 h transfection, the cells were treated with 5 mM HU for 5 h. The protein levels of FANCM and FANCD2 were measured by western blotting. Monoubiquitinated FANCD2 is indicated as the upper band of doublet protein bands corresponding to FANCD2. (C and D) After HeLa cells underwent the same treatment as (A and B) cells were analyzed for FANCD2 foci formation. DAPI was used for nuclear staining. Results are shown as the mean ± SD (n = 3); **P < 0.01. (E and F) Indicated cells were treated with 5 mM HU for 5 hr. Cell lysates were analyzed by Western blotting with antibodies against pCHK1-S317, CHK1, pRPA-S4/8, and RPA.

Fig 3: Knockdown of PIG3 changes the DNA damage response and cell cycle progression of HeLa cells. (A) The phosphorylation of Chk1, Chk2 and H2AX was detected by immunoblotting analysis at various time points after 4 Gy of ?-ray irradiation in PIG3-depleted (HeLa-shPIG3) and control (HeLa-shNC) cells. (B, C) Cell cycle distribution of PIG3-depleted cells and control cells was detected by Flow cytometry assay after 4 Gy of ?- irradiation. (D) HeLa PIG3-depleted cells and control cells were synchronized in G1 phase by TdR double blocking method and then released, and cell cycle distributions at the indicated time points were examined. (E) 5×103 cells were plated at day 0 and cell numbers were counted every day to generate a growth curve. The data are the mean ± S.D. from three tests. (F) HeLa PIG3-depleted cells and control cells were synchronized at G1 phase by TdR double blocking method and then released, the p53 and cyclin B1 levels at the indicated times were examined by immunoblotting.

Fig 4: DNA damage response and DNA-PKcs expression in PIG3-depleted cells after a number of cell cycles or passages of subculture. (A) The phosphorylation and protein level of a number of DDR proteins were detected by immunoblotting analysis at various time points post 8 Gy of ?-irradiation in PIG3-depleted (MCF7-shPIG3) and control (MCF7-shNC) cells. (B) The level of p-Chk2 T68 and p-Chk1 S317 was detected by immunoblotting analysis at various time points after 10 J / m2 of UV irradiation in PIG3-depleted (MCF7-shPIG3) and control (MCF7-shNC) cells. (C) The DNA-PK complex was detected in HeLa cells after more than 5 passages of subculture after PIG3 knockdown. (D) HeLa cells were infected with the viral particles expressing PIG3 shRNA, and 48 hours later puromycin was added into the medium to kill uninfected cells. At 7 days post-infection, cells stopped dying and were collected at the indicated days. PIG3 and DNA-PKcs levels were detected by SDS-PAGE. (E) Human fibroblast cells HFS were infected by the viral particles of expressing PIG3 shRNA. PIG3 and DNA-PKcs levels were tested at the indicated days post infection.