Fig 2: Overexpression of CTDP1 regulates FANCI SQ motif phosphorylation at S556 and S559 and chromatin localization.a FANCI SQ motif phosphorylation at S556 (pS556) and activation of ATM and ATR was evaluated with the indicated antibodies in 293FT cells transfected with control empty vector (EV) or CTDP1-DDK-tagged constructs in the presence and absence of DNA damage with 0.1 µM MMC for 16 h. b FANCI SQ motif phosphorylation at S556 (pS556) upon CTDP1 knockdown and MMC treatment. c Wild-type (WT), phosphatase-dead (D302K), and cancer mutants (R270Q and V705M) constructs of CTDP1 were expressed in 293FT cells treated with MMC, and the impact on SQ motif phosphorylation sites at S556 and S559 were evaluated by western blot with the indicated antibodies. d FANCI phosphorylation at S556 was evaluated with and without 0.4 µM MMC in MDA-MB-231 cells transfected with either EV or DDK-CTDP1 expression constructs. e shScr and shCTDP1 HCT116 cells were treated with or without 100 µM melphalan for 6 h and subjected to cellular fractionation and both CTDP1 and FANCI localization to chromatin was determined by western blot. Total H2AX and tubulin are included as fractionation control for chromatin and soluble protein fractions. Short exposure (S.E.). Long exposure (L.E.) f FANCI association with the DNA damage marker ?-H2AX was evaluated by immunoprecipitation of FANCI (IP:FANCI) followed by western blot for ?-H2AX 293FT cells either untreated or treated with 50 µM cisplatin for 16 h. Dashed line in the CTDP1 blot indicates the image has only been modified to maintain the correct order of samples

Fig 3: Melinjo resveratrol and chlorogenic acid contribute to genome stability maintenance. (a) The immortalisation of MEFs cultivated under the Std-3T3, MelRes-3T3 (melinjo resveratrol) or Chlo-3T3 (chlorogenic acid) protocol as indicated. In the MelRes-3T3 and Chlo-3T3 protocols, MEFs were regularly treated with 0.5 µg/ml melinjo resveratrol and 2.5 µM chlorogenic acid, respectively. In the lower graph, data are represented as the mean ± s.d. (n = 3 biologically independent experiments). (b) The effects of cultivation of MEFs under the Std-3T3, MelRes-3T3 or Chlo-3T3 protocol on chromosomal instability (tetraploidy). (c,d) Time- and dose-dependent effects of melinjo resveratrol (c) and chlorogenic acid (d) on H2AX expression in senescent MEFs (passage 8). The dose-dependent effects were assessed 3 hr after treatment. The time-dependent effects were determined using 0.5 µg/ml melinjo resveratrol (c) or 5 µM chlorogenic acid (d). (e) The effects of melinjo resveratrol (0.5 µg/ml) and chlorogenic acid (5 µM) on the number of ?H2AX foci in senescent MEFs at 3 and 24 hr post-treatment. ?H2AX foci were detected by immunofluorescence (n numbers are indicated in the graphs). Data in the graphs are represented as the mean ± s.d. Scale bars, 10 µm. P-values were calculated by two-tailed Welch’s t-tests.

Fig 4: Regular resveratrol treatment maintains genome stability and suppresses DSB accumulation. (a) The immortalisation of MEFs cultivated under the Std-3T3 or Resv-3T3 protocol as indicated. In the Resv-3T3 protocol, the cells were treated with 2.5 µM resveratrol regularly. In the lower graph, data are represented as the mean ± s.d. (n = 3 biologically independent experiments). (b) The effects of cultivation of MEFs under the Std-3T3 or Resv-3T3 protocol on chromosomal instability (tetraploidy). (c) Statuses of the ?H2AX and 53BP1 foci in MEFs at a growth-arrested stage (P8 + 3 days) following cultivation under the Std-3T3 or Resv-3T3 protocol. The numbers of ?H2AX foci were quantified and data are represented as the mean ± s.d. (n = 3 biologically independent experiments). (d,e) Immunostaining (d) and immunoblot (e) analyses of H2AX in quiescent MEFs grown under the Std-3T3 or Resv-3T3 protocol. (c,d) Scale bars, 10 µm.

Fig 5: CTDP1 knockdown impairs FANCA and FANCD2 DNA damage-induced foci.a Western blot demonstrating knockdown of CTDP1 and FANCA using targeted shRNAs in MCF-10A cells. b Representative immunofluorescence of FANCA alone and merged with DAPI (white and blue color, respectively) in MCF-10A cells expressing the indicated shRNAs (shScr, shCTDP1, or shFANCA) treated with 0.2 µM MMC for 24 h. Scale = 5 µm. c Graph representing the percentage of nuclei containing = 5 FANCA foci with or without MMC treatment. n = 3 independent experiments; mean ± SEM; paired two-tailed Student’s t-test; =200 cells evaluated/experiment. d Representative immunofluorescence of FANCD2 alone and merged with DAPI (white and blue color, respectively) in MCF-10A cells expressing the indicated shRNAs (shScr, shCTDP1, or shFANCA) treated with 0.2 µM MMC for 24 h. Scale = 5 µm. e Graph representing the percentage of nuclei containing = 5 FANCD2 foci with or without MMC treatment. n = 3 independent experiments; mean ± SEM; paired two-tailed Student’s t-test; = 100 cells evaluated/experiment. f Representative immunofluorescence of ?-H2AX alone and merged with DAPI (white and blue color, respectively) in MCF-10A cells expressing the indicated shRNAs (shScr, shCTDP1, or shFANCA) treated with 0.2 µM MMC for 24 h. Scale = 5 µm. g Graph representing the percentage of nuclei containing = 5 ?-H2AX foci with or without MMC treatment. n = 3 independent experiments; mean ± SEM; paired two-tailed Student’s t-test; = 200 cells evaluated/experiment. h Analysis of FANCD2 mono-ubiquitination using cellular fractionation of MCF-10A cells (shScr and shCTDP1) with and without MMC treatment for 24 h by calculating the ratio of long (mono-ubiquitinated): short (non-ubiquitinated) proteoforms quantified by LI-COR signal intensities. (S, soluble protein fraction, including cytosol and nucleoplasm; P, pellet protein fraction, contains chromatin)