Fig. 4

The XRCC1 KD-induced activation of the ISR is mediated through signalling of persistent single-stranded DNA damage via ATM. a Alkaline comet assay of siControl and siXRCC1 cells. b–d Representative Western blots of b PAR and XRCC1 after siXRCC1. The smear above 100 kDa marker indicates PAR. c pATM and ATM after siXRCC1 using 3 different siRNA sequences. d γH2AX, Histone H3, XRCC1, and Tubulin in cells after siXRCC1, or 4 Gy IR. e Neutral comet assay of cells treated after siControl, siXRCC1, or 4 Gy IR. n = 4. f, g Quantification of 53 bp1 (f) or γH2AX (g) foci per nucleus in cells after 4 Gy IR, siControl, or siXRCC1. h–j Representative Western blots of h ATF4 in siControl or siXRCC1 cells treated with Ly or DMSO; i ATF4 in siControl or siXRCC1 cells treated with KU, Olap, or DMSO; j ATF4 in siControl or siXRCC1 cells treated with ATMi or DMSO; and k peIF2a in siControl or siXRCC1 cells treated with ATMi or DMSO. Normalised levels of ATF4 or peIF2α are shown below the lanes. l–o Phase-contrast images of siControl (l, m) or siXRCC1 (n, o) cells treated with DMSO (l, n) or ATMi (m, o), grown in a medium containing 1% FCS. Images are from one representative experiment, with four different fields randomly chosen per condition. Scale bar = 400 μm. p Quantification of relative cell area as shown in l–o normalised to siControl + DMSO grown at 1% FCS. n = 3. For all quantifications, only statistically significant differences are indicated using asterisks