Fig. 3: TEX264 counteracts TOP1ccs.

a Immunofluorescent detection of TOP1ccs (green) in RPE-1 cells transfected with the indicated siRNAs. DAPI 4′,6-diamidino-2-phenylindole. Scale bar, 10 µm. b Quantification of a. Red line indicates median. Data are representative of two independent experiments. Significance determined by Mann–Whitney test. c Slot blot analysis of TOP1ccs prepared from WT or ΔTEX264 HEK293 cells using the RADAR assay. d Quantification of c (error bars represent mean ± SD; n = 2; **P < 0.01, *P < 0.05; Student’s t-test). e Representative images of cells analysed by alkaline comet assay in f. f Alkaline comet assay performed in RPE-1 cells treated with the indicated combinations of siRNAs. Cells were treated with CPT (100 nM) for 1 or 6 h(s) and allowed to recover as indicated. Quantification of >100 cells per condition from one representative experiment is shown, and the experiment was repeated two times. Whisker box plots show median values and data within the 10–90 percentile. Statistical analysis was performed using Kruskal–Wallis ANOVA with multiple comparisons, with Benjamini–Hochberg post-test. g Colony forming assay to assess the viability of HeLa cells transfected with the indicated siRNAs. Cells were treated for 24 h with the indicated doses of CPT, released into normal media for 7 days, then fixed and stained. Viability represents the number of colonies in each sample expressed as a percentage of the number of colonies formed in the corresponding untreated sample (error bars represent mean ± SD; n = 2). h Schematic diagram of the TEX264 protein indicating the location of residue E194. i Left: Immunoblots of FLAG immunoprecipitates prepared from ΔTEX264 HEK293 cells transiently expressing the indicated versions of FLAG-tagged TEX264. Right: Corresponding quantifications of four independent experiments (mean ± SEM; ****P < 0.0001; ***P < 0.0005; Student’s t-test). Source data are available online.