Fig 2: TFIIH integrity analysis in strains expressing Tfb2 HubA mutants.A, whole-cell extracts from Tfb6+ strains expressing the indicated FLAG-tagged Tfb2 derivatives and pA-tagged Rad3 were subjected to IP and Western analysis using antisera against the indicated TFIIH subunits. Extracts prepared from a strain expressing untagged Rad3 and FLAG-tagged WT Tfb2 were used as a control. B, quantitation of Western analysis. Significantly affected subunits are indicated by ∗ (padj <0.04). Red dots indicate data that correspond to the Western blots shown in A. C, whole-cell extracts from Δtfb6 strains expressing the indicated FLAG-tagged Tfb2 derivatives and pA-tagged Rad3 were subjected to IP and Western analysis using antisera against the indicated TFIIH subunits. Extracts prepared from a strain expressing untagged Rad3 and FLAG-tagged WT Tfb2 were used as a control. D, quantitation of Western analysis. Significantly affected subunits are indicated by ∗ (padj <0.04). IP, immunoprecipitation; padj, adjusted p value.

Fig 3: Effects of XPD overexpression on multiple signaling pathways in EC9706 cells and EC109 cells. (A and B) The mRNA expression levels of PI3K, AKT, Bcl-2, c-Myc, Cyclin D1, VEGF, MMP-9 and p21 in EC9706 or EC109 cells were detected by RT-qPCR. *P<0.05 or **P<0.01, vs. Ctrl group or pEGFP-N2 group (n=3). (C-F) The protein expression levels of PI3K, p-AKT, AKT, Bcl-2 and p21 in EC9706 or EC109 cells were detected by western blot analysis. (G and H) Following treatment with or without LY294002 (10 µM), cell proliferation was examined by CCK-8 assay. *P<0.05 or **P<0.01, vs. pEGFP-N2/XPD group (n=3). (I and J) The protein expression levels of p-p65, p65, p-STAT3, STAT3, p-p38, and p38 in EC9706 cells were detected by western blot analysis. Data represent the means ± standard deviation. pEGFP-N2/XPD, pEGFP-N2/XPD plasmid was transfected into EC9706 or EC109 cells; pEGFP-N2, pEGFP-N2 plasmid was transfected into EC9706 or EC109 cells; Ctrl, no transfection; pEGFP-N2/XPD + LY294002, pEGFP-N2/XPD plasmid was transfected into EC9706 or EC109 cells and cells were then treated with LY294002 (10 µM); XPD, xeroderma pigmentosum complementation group D.

Fig 4: Upregulation of XPD enhances the chemosensitivity of EC9706 and EC109 cells to cisplatin or fluorouracil. The effects of XPD upregulation on the chemosensitivity of EC9706 and EC109 cells to (A and B) cisplatin or (C and D) fluorouracil were determined by CCK-8 assay. Date represent the means ± standard deviation. *P<0.05 or **P<0.01, vs. Ctrl group or pEGFP-N2 group (n=3). pEGFP-N2/XPD, pEGFP-N2/XPD plasmid was transfected into EC9706 or EC109 cells; pEGFP-N2, pEGFP-N2 plasmid was transfected into EC9706 or EC109 cells; Ctrl, no transfection; XPD, xeroderma pigmentosum complementation group D.

Fig 5: XPC and TFIIH are continuously recruited to UV damage in XPCS patient fibroblasts. (A) Representative immunofluorescence pictures showing the co-localization of XPF and XPB at LUD in patient fibroblasts XPCS1CD and CS1USAU, compared to wild type C5RO fibroblasts. Cells were stained 1 h and 8 h after local UV irradiation (60 J/m2) through an 8 μm microporous filter with antibodies against XPF and XPB. (B) Representative immunofluorescence pictures showing the LUD recruitment of XPD and XPC in fibroblasts derived from patients carrying mutations in XPB, XPD, XPG, XPF and XPA, affected by mild XP (XP42RO, XP32BR, XP6BE), XPCS (XPCS1CD, XPCS1BA, XPCS2, XPCS1RO) or DSC (XP25RO) compared to wild type C5RO fibroblasts. Cells were locally UV irradiated with 60 J/m2 UVC through an 8 μm microporous filter and stained 1 h and 8 h after irradiation with antibodies against XPD and XPC. Scale bar: 5 μm.