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. 2021 Oct 30;49(20):11746–11764. doi: 10.1093/nar/gkab999

Figure 5.

Figure 5.

PCNA accumulates on DNA upon oxidative damage in ATAD5-depleted cells at G1 phase. (A) HeLa-ATAD5mNeonGreen-AID cells were transfected with ATAD5 siRNA targeting 3′UTR and subjected to 405-nm UV laser microirradiation. Two minutes after microirradiation, cells were fixed for PCNA immunostaining; scale bar: 10 μm. (B–F) Asynchronous U2OS-ATAD5AID cells (B and C) or U2OS-ATAD5AID cells G1-enriched by treatment with PD 0332991 and PHA-767491 (D–F) were treated with auxin for 16 h and then treated with H2O2 as indicated. (B–E) After H2O2 treatment, cells were detergent-pre-extracted, fixed and immunostained with anti-PCNA antibody. (B and D) Representative images of PCNA immunostaining; scale bar: 20 μm. (C and E) Quantification of chromatin-bound PCNA signal intensity. Three independent experiments were performed and one representative result is displayed. Red bar indicates mean value. Statistical analysis: two-tailed unpaired Student’s t-test; ****P < 0.001. (G and H) U2OS cells (G) or HeLa cells (H) transfected with ATAD5 siRNAs were enriched at G1 phase by treatment with PD 0332991 and PHA-767491 (G) or by releasing cells from nocodazole arrest (H), and then treated with 0.5 mM H2O2 for 1 h. #1 and #3: siRNAs targeting an exon region of ATAD5 gene. (F–H) After H2O2 treatment, detergent-insoluble proteins were isolated and subjected to immunoblotting.