Journal: Nucleic Acids Research

Article Title: REV1 and polymerase ζ facilitate homologous recombination repair

doi: 10.1093/nar/gkr769

Figure Lengend Snippet: REV1 and Polζ promote radioresistance independently of RAD18. ( A and B ) HeLa cells transfected with siRNAs targeting RAD18 or RAD51 were exposed to 4 J/m 2 UV-C light or 4 Gy IR as indicated. Twenty-four hours later, cells were subjected to cell cycle analysis by flow cytometry to measure DNA content per cell. ( C ) HeLa cells depleted of RAD18 stain intensely for γ-H2AX 24 h after exposure to UV-C irradiation indicative of strong DNA damage response due to replication fork stalling and efficient protein knockdown. ( D ) RAD18-depleted HeLa cells are proficient in resolving foci containing S1981P-ATM and 53BP1 within 24 h. Cells transfected with RAD51 siRNA were analyzed as a positive control for HR repair deficiency. The mean percentage of cells exhibiting 10 or more co-localized foci is shown in ( E ). Data are the mean ± SEM of three independent experiments.

Article Snippet: Anti-MAD2L2 (REV7) and anti-RAD18 polyclonal antibodies were obtained through Proteintech Group.

Techniques: Transfection, Cell Cycle Assay, Flow Cytometry, Staining, Irradiation, Knockdown, Positive Control

Journal: Life Science Alliance

Article Title: RFWD3 and translesion DNA polymerases contribute to PCNA modification–dependent DNA damage tolerance

doi: 10.26508/lsa.202201584

Figure Lengend Snippet: (A) WI38VA13 cells were transfected with siRNA against REV1 (siREV1), REV7 (siREV7), Polκ (siPolκ pool or siPolκ#2), Polι (siPolι), or non-targeting control siRNA (siNTC#1). Transfected cells were exposed to the indicated doses of illudin S for 4 d. Cellular survival was evaluated by MTS assay. Data are represented as means ± SD of n = 6 (siNTC#1 and siREV1), n = 4 (siPolκ pool and siPolκ#2), or n = 3 (siREV7, and siPolι) independent experiments. The data for PCNA[KR] are replotted from . (B) Whole-cell lysates were prepared from the cells used in (A) and analyzed by immunoblotting using anti-REV1, anti-REV7, anti-Polι, anti-Polκ, and anti-Lamin B antibodies. (C) HeLaS3, HeLaS3 Polκ KO, and HeLaS3 Polκ KO/GFP-Polκ cells were exposed to illudin S for 4 d. Cellular survival was evaluated by MTS assay. Data are represented as means ± SD of n = 7 (HeLaS3) or n = 3 (HeLaS3 Polκ KO and HeLaS3 Polκ KO/GFP-Polκ) independent experiments. (D) Whole-cell lysates were prepared from the cells used in (C) and analyzed by immunoblotting using anti-Polκ and anti-Lamin B antibodies. (E) WI38VA13 cells were transfected with siRNAs against RFWD3 (siRFWD3#1 or siRFWD3#2), siRFWD3#1+siPolκ#2, or non-targeting control siRNA (siNTC#2 or siNTC#3). Transfected cells were exposed to illudin S for 4 d and cellular survival was evaluated by MTS assay. Data are represented as means ± SD of n = 7 (siRFWD3#1), n = 6 (siNTC#2), or n = 3 (siNTC#3 and siRFWD3#2) independent experiments. The data for PCNA[KR] and siPolκ#2 are replotted from and , respectively. (F) Whole-cell lysates were prepared from the cells used in (E) and analyzed by immunoblotting using anti-RFWD3 and anti-Lamin B antibodies. The arrowhead shows the RFWD3 signal. (G) CS1ANSV cells were transfected with siNTC#2, siRFWD3#1, or siPolκ#2. Transfected cells were exposed to illudin S for 4 d and cellular survival was evaluated by MTS assay. Data are represented as means ± SD of n = 3 independent experiments. (H, I) WI38VA13 cells were transfected with siRFWD3#1, Polκ#2, siRFWD3#1+siPolκ#2, or NTC#2. Cells were exposed to 25 ng/ml illudin S and 20 μM BrdU for 1 h and incubated for indicated periods without the drugs. Cells were analyzed as described in . (H) FACS profiles. (I) The proportion of BrdU-positive S-phase cells was calculated. Data are represented as mean ± SD of n = 3 independent experiments. (J) WI38VA13 cells were transfected with siRFWD3#1, siPolκ#2, or siNTC#2. Cells were exposed to 25 ng/ml illudin S for 1 h and then incubated for the indicated periods without the drug. Cells were treated with 20 μM BrdU for 1 h at the indicated time points, harvested, and fixed. FACS analyses were performed as described in . BrdU intensities are shown. (−), untreated sample. Dotted lines show the median intensity of incorporated BrdU in untreated cells. (K) WI38VA13 cells were transfected with siRFWD3#1, Polκ#2, siRFWD3#1+siPolκ#2, or NTC#2. Cells were labeled with 25 μM CldU for 30 min, exposed to 50 ng/ml illudin S for 1 h, incubated for 3 h without the drug, and labeled with 250 μM IdU for 30 min. Incorporated CldU and IdU were stained with anti-BrdU antibodies. Quantified CldU (red) and IdU (green) track length were shown. At least 100 tracks from two independent experiments were evaluated. The line represents the median; boxes are the 25th and 75th percentiles; whiskers are the minimum and the maximum values. (L, M, N) siRFWD3#1, siPolκ#2, or siNTC#2-transfected WI38VA13 cells were exposed to 25 ng/ml illudin S for 1 h and incubated for 6 or 18 h without the drug. RPA and γH2AX were detected and quantified as described in . (L) Representative images. Scale bar represents 20 μm. (M, N) Quantified RPA (M) or γH2AX (N) intensities in each nucleus. At least 150 nuclei were evaluated. (O) WI38VA13 cells were transfected with siRFWD3#1, siPolκ#2, or siNTC#2, exposed to 25 ng/ml illudin S for 1 h, and incubated for the indicated periods without the drug. (−), untreated with illudin S. Whole-cell lysates were prepared and analyzed by immunoblotting using anti-phospho RPA2 (S4/S8), anti-RPA2, anti-γH2AX, and anti-H2AX antibodies. The statistical significance was evaluated by two-tailed t tests. ns, not significant. Source data are available for this figure.

Article Snippet: The primary antibodies used were as follows: rabbit anti-RAD18 (1:2,000; 70-023; Bio Academia), mouse anti-PCNA (PC10) (1:10,000 or 1:2,000; sc-56; Santa Cruz Biotechnology), goat anti-Lamin B (C-20) (1:3,000; sc-6216; Santa Cruz Biotechnology), mouse anti-SHPRH (3F8) (1:2,000; TA501443; ORIGENE), rabbit anti-HLTF (1:3,000; ab17984; Abcam), rabbit anti-XPA (FL-273) (1:3,000; sc-853; Santa Cruz Biotechnology), goat anti-CSB (E-18) (1:3,000; sc-10459; Santa Cruz Biotechnology), rabbit anti-XPC (1:2,000) , guinea pig anti-REV1 (1:10,000) , mouse anti-Polκ (A-9) (1:2,000; sc-166667; Santa Cruz Biotechnology), mouse anti-REV7 (1:2,000; 612266; BD Biosciences), rabbit anti-Polι (1:1,000; ab123331; Abcam), mouse anti-FANCD2 (FI17) (1:2,000; sc-20022; Santa Cruz Biotechnology), rabbit anti-BRCA1 (C20) (1:3,000; sc-642; Santa Cruz Biotechnology), mouse anti-RPA2 (1:2,000; ab2175; Abcam), mouse anti-γH2AX (1:2,000; 05-636; Merck Millipore), rabbit anti-phospho RPA2 (S4/S8) (1:2,000; A300-245A; Bethyl Laboratories), rabbit anti-H2AX (1:2,000; ab11175; Abcam), rabbit anti-Lamin B1 (1:10,000; 12987-1-AP; Proteintech), mouse anti-β-actin (6D) (1:10,000; M177-3; Medical & Biological Laboratories), and rabbit anti-ubiquityl PCNA (Lys164) (D5C7P) (1:1,000; 13439; Cell Signaling Technology).

Techniques: Transfection, MTS Assay, Western Blot, Incubation, Labeling, Staining, Two Tailed Test