Journal: Nature Communications

Article Title: UBE2D3 facilitates NHEJ by orchestrating ATM signalling through multi-level control of RNF168

doi: 10.1038/s41467-024-49431-6

Figure Lengend Snippet: a Immunoblotting for pATM, γH2AX and CHK2 in TRF2ts MEFs transduced as indicated and upon telomere uncapping at 39 °C. Asterisk indicates ATM phosphorylated at Ser1981. Representative blots from 4 independent experiments. b Quantification of pATM, γH2AX, FK2 and RIF1 foci in TRF2ts MEFs, transduced as indicated, at 32 °C (0 h) or upon telomere uncapping for 3 h at 39 °C ( n = 3 independent experiments for pATM and RIF1, n = 6 independent experiments for γH2AX and n = 4 independent experiments for FK2; mean ± SEM), and quantification of 53BP1 foci at telomeres (telomere dysfunction-induced foci (TIFs)) in TRF2ts MEFs upon telomere uncapping for 3 h at 39 °C ( n = 5 independent experiments; mean ± SEM). Statistical significance was calculated using the two-tailed Student’s t -test. c Immunoblotting for RNF8, 53BP1, RIF1 and MAD2L2 in TRF2ts MEFs subjected to telomere uncapping at 39 °C. Representative blots from 4 independent experiments (RNF8 and 53BP1) or 2 independent experiments (MAD2L2 and RIF1). The asterisk indicates 53BP1; below is a non-specific band. d RNF168 levels in TRF2ts MEFs upon telomere uncapping. Representative blots from 5 independent experiments. e Quantification of RNF168 levels in whole cell extracts (WCE) ( n = 5 independent experiments; mean ± SEM; one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test). f Immunoblots for GFP-RNF168 in control and UBE2D3-depleted TRF2ts MEFs. Asterisk indicates endogenous RNF168. Representative blots from 4 independent experiments (quantifications in Supplementary Fig. ). g Quantification of RNF168 protein levels in HEK 293T cells with complementation of UBE2D3 depletion by RNAi-resistant (RR) wild-type UBE2D3 (pCDH_UBE2D3_WT_RR) or UBE2D3 C85A (pCDH_UBE2D3_C85A_RR) ( n = 4 independent experiments; mean ± SEM; one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test). Representative immunoblots in Supplementary Fig. . h Immunoblotting of chromatin fractions to assess RNF168 recruitment to chromatin upon telomere uncapping at 39 °C. Representative blots from 3 independent experiments. i Quantification of GFP-RNF168 foci in TRF2ts MEFs at 32 °C and upon 3 h of telomere uncapping at 39 °C. Ref reference. Source data are provided as a file.

Article Snippet: Primary antibodies used were against UBE2D3 (Y-25, sc-100618, SCBT, 1:500; 11677-1-AP, Proteintech, 1:500; 4330S, CST, 1:500 and A615, Boston Biochem, 1:2000), KAP1 (22553, Abcam, 1:1000), phospho-Kap1 S824 (A300 767A, Bethyl, 1:1000), 53BP1 (NB100-305, Novus, 1:500 and A300-272A, Bethyl, 1:2000), phospho-ATMS1981 (4526, CST, 1:1000), phospho-H2AX S139 (5636, Millipore, 1:1000), CHK2 (611570, BD, 1:500), c-myc (9E10, sc-40, SCBT, 1:250), HA (MMS-101R, Covance, 1:1000), TRF2 (NB110-57130, Novus, 1:500), RNF8 (sc-133971, SCBT, 1:250), MAD2L2 (14, sc-135977, SCBT, 1:500), GFP IgG fraction (A11122, Thermo Fisher Scientific, 1:1000), FLAG M2 (F1804, Sigma-Aldrich, 1:1000), Histone H3 (ab1791, Abcam, 1:10,000), hRNF168 (ABE367, Millipore, 1:500), hRNF168 (ABE467, Merck-Millipore, 1:1000), mRnf168 (gift from D. Durocher, 1:1000), hRIF1 (A300-569A, Bethyl, 1:1000), mRIF1 (gift from S. Boulton and R. Chapman, 1:1000), Ligase 4 (H-300, sc-28232, SCBT, 1:300; NB110-57379, Novus, 1:500), FK2 (04-263, Millipore, 1:2000), HP1α (2616S, CST, 1:1000), Ubiquitin (P4D1, sc-8017, SCBT, 1:1000), HDAC1 (PA1-860, Thermo Fisher Scientific, 1:1000), PP2A C subunit, clone 1D6 antibody (05-421, Sigma-Aldrich/Millipore, 1:500), CDK4 (C-22, sc-260, SCBT, 1:500), HSP90 α/β (H-114, sc-7947, SCBT, 1:1000), γ-tubulin (T6557, Sigma-Aldrich, 1:10,000) β-actin (A5316, Sigma-Aldrich, 1:10,000), β-catenin (610154, BD, 1:10,000) and GAPDH (PA1-987, Thermo Fisher Scientific, 1:1000).

Techniques: Western Blot, Two Tailed Test, 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