Fig 1: Interactions of DONSON and FANCM with replisomes and chromatin in nontreated cells.a DONSON associates with some, but not all, replisomes in untreated cells. Chromatin was prepared from untreated GFP-DONSON-HeLa cells and sequential IP performed, first against GFP-DONSON, and then against the GINS protein PSF1 from the residual supernatant. Representative blot (n = 3). b PLA of GFP-DONSON and PSF1 demonstrates DONSON-associated replisomes are more frequent in early S phase than in late S phase in NT cells. Scored nuclei: PLA between GFP-D: PSF1 early S phase = 82, late S phase = 81, from three biological replicates. Data are mean ± s.e.m. c PLA between FANCM and MCM2 demonstrates low level of FANCM-associated replisomes in late S phase in nontreated cells. Scored nuclei: PLA between FANCM: MCM2 of early S phase = 73, late S phase = 75, from three biological replicates. Data are mean ± s.e.m. d IP of FANCM demonstrates low-level interaction with replisome protein MCM2. e PLA between GFP-DONSON and H3K4me3 or H3K9me3. Scored nuclei of GFP-DONSON and H3K4me3 in early S phase = 79, late S phase = 78. Scored nuclei of GFP-DONSON and H3K9me3 in early S phase = 77, late S phase = 82, from three biological replicates. Data are mean ± s.e.m. f PLA between FANCM and H3K4me3 or H3K9me3. Scored nuclei of FANCM and H3K4me3 in early S phase = 64, late S phase = 65. Scored nuclei of FANCM and H3K9me3 in early S phase = 68, late S phase = 65, from three biological replicates. Data are mean ± s.e.m. For PLA experiments in b, c, e, f, a two-sided Mann–Whitney rank-sum test was used to determine if differences were statistically significant. NS: not significant: P > 0.05. Source data are provided as a Source Data file.
Fig 2: DONSON and FANCM operate in separate pathways to promote replication traverse.a HeLa cells were treated with siRNA against DONSON or FANCM or both. They were exposed to Dig-TMP/UVA and incubated with CldU, then IdU. Fibers were prepared and patterns displayed by immunofluorescence against the analogues and immunoquantum detection (Q dot 655, in red) for Dig-tagged ICLs. Representative patterns are shown. b Quantitation of pattern distribution. Fibers with ICL encounters: NT = 417; siDONSON = 432; siFANCM = 417; siDONSON + siFANCM = 385, from three independent replicates. Data are mean ± s.d. c IP immunoblot of chromatin proteins from cells expressing GFP (panels 1, 2) or GFP-DONSON (panels 3, 4) exposed to UVA (-) or TMP/UVA (+). The identity of the proteins is indicated. The amounts of PSF1 and CDC45 in the two samples were quantitated. Representative blot (n = 3). Data are mean ± s.d. d PLA test of the influence of ATR inhibition on GFP-DONSON interactions with pMCM2S108, MCM2, and MCM5. Number of nuclei: PLA between GFP-DONSON and pMCM2 in cells treated with UVA = 58, TMP/UVA = 94, TMP/UVA + ATRi = 55; PLA between GFP-DONSON and MCM2 in UVA = 95, TMP/UVA = 89, TMP/UVA + ATRi = 93; PLA between GFP-DONSON and MCM5 in UVA = 88, TMP/UVA = 79, TMP/UVA+ATRi = 73; from three biological replicates. Data are mean ± s.e.m. e PLA assessing the influence of ATR inhibition on GFP-DONSON interactions with CDC45 and PSF1. Scored nuclei of PLA between GFP-DONSON and CDC45 in UVA = 70, TMP/UVA = 71, TMP/UVA + ATRi = 73; scored nuclei of PLA between GFP-DONSON and PSF1 in UVA = 71, TMP/UVA = 64, TMP/UVA + ATRi = 77; from three biological replicates. Data are mean ± s.e.m. f Influence of ATR inhibition on the PLA between GFP-DONSON and Dig-tagged ICLs. Scored nuclei: Vehicle = 72, ATRi = 87, three biological replicates. Data are mean ± s.e.m. For replication pattern frequency experiments and Western blotting image analysis (a, c), a two-sided unpaired t test was used to calculate P-values. For PLA experiments (d–f), a two-sided Mann–Whitney rank-sum test was used to determine if differences were significant. NS: not significant: P > 0.05. Source data are provided as a Source Data file.
Fig 3: Antitumor efficacy of Arbidol in an ESCC patient-derived xenograft model.(a) Arbidol treatment protocol for esophageal squamous cell carcinoma (ESCC) patient-derived xenograft (PDX) models. (b) Tumor sizes of the EG20 xenografts were shown. Tumors were excised and weighed at the end of the experiment (36 days after treatment). (c) The effect of Arbidol inhibition on in vivo tumor growth was determined using ESCC PDX models: EG20. Mice (n=7 per group) were treated with vehicle (saline, oral administration, daily) or Arbidol (low = 100 mg/kg, high = 400 mg/kg, daily) for 36 days. Tumor volumes were measured every 3 days. Data were analyzed by homogeneity of variance and one-way analysis of variance (ANOVA). Data are presented as the mean, ****p<0.0001. (d) Tumor growth curve of single mouse grafted with EG20 is shown. n=7 per group. (e) Weight of mice treated with Arbidol. Data were analyzed by homogeneity of variance and one-way analysis of variance (ANOVA). *p<0.05, ***p<0.001. (f) Tumor volume of each group was shown, respectively, n=7 per group. (g) The expression of tumor proliferation markers Ki67 and target engagement was verified by immunohistochemical analysis of minichromosome maintenance 2 (MCM2) S108 expression in Arbidol-treated EG20 PDX mice, n=7 per group. Data were analyzed by homogeneity of variance and one-way ANOVA. *p<0.05, ***p<0.001, ****p<0.0001. Scale Bars: 50 µm.
Fig 4: The validation results and changes of cell cycle signal molecules after CCT3 silencing in melanoma cells. (A) qRT-PCR showed the upregulation of 12 genes and downregulation of two genes in CCT3-KD A375 cells. (t test, P < 0.01 for all genes). (B) and (C). Western blotting analysis showed decreased levels of CDK1, FOXM1, MCM2, PIM1, and SKP2 genes and no statistical difference for NFKBIA in CCT3-KD A375 cells. (D). RT-qPCR results indicated that the expression of CDK1 mRNA was successfully rescued after shCCT3 melanoma cells were infected with CDK1 overexpression lentivirus vector (OE-CDK1). (E) MTT assay showed that decreased cell proliferation by CCT3 silencing was rescued by the overexpression of CDK1. *P < 0.05, **P < 0.01, ***P < 0.001, compared with the shCtrl cells.
Fig 5: DONSON and FANCM are on different replisomes.a Scheme of sequential IP against DONSON and FANCM-associated replisomes. HeLa cells expressing GFP-DONSON were exposed to UVA only or TMP/UVA. Chromatin was prepared and digested with benzonase. This was followed by IP against PSF1 (to remove unstressed replisomes), then IP of the supernatant against GFP (to remove remaining DONSON-associated proteins), and finally IP of the residual supernatant to capture FANCM-bound proteins. b Western blot analysis of sequential IP. Representative blot (n = 3). c PLA in cells exposed to UVA only or TMP/UVA shows interactions between GFP-DONSON and MCM2; and FANCM and MCM2; but not between GFP-DONSON and FANCM. Scored nuclei: PLA between GFP-D: MCM2, UVA treatment = 174; TMP/UVA = 148; PLA between FANCM: MCM2, UVA = 142; TMP/UVA = 145; PLA between GFP-D: FANCM, UVA = 135; TMP/UVA = 133 from three biological replicates. Data are mean ± s.e.m. A two-sided Mann–Whitney rank-sum test was used to determine if differences were significant. NS: not significant: P > 0.05. d Association of replisomes with Dig-tagged ICLs. Chromatin was prepared from cells exposed to UVA or Dig-TMP/UVA, and the DNA reduced to fragments of <500 bp by sonication. Sequential IP was performed, and the DNA isolated from each fraction, dotted onto the nitrocellulose, and probed with an antibody to the Dig tag. LINE-1 repeat element served as a loading control. Representative blot (n = 2). e Model summarizing the results of the sequential IP experiment. Source data are provided as a Source Data file.
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