Fig. 6. TEX264 acts at replication forks.
a Immunoblots of anti-Strep-tag immunoprecipitates prepared from doxycycline (Dox)-inducible TEX264-SSH HEK293 Flp-In TRex cells, incubated with and without Dox. b Measurement of EdU incorporation rates in four different cell lines treated with the indicated siRNAs. EdU incorporation is plotted as a percentage of the corresponding control cells. Cells were incubated with 10 µM EdU for 30 min prior to collection (error bars represent mean ± SEM; ****P < 0.0001; ***P < 0.0005; **P < 0.01, *P < 0.05; Student’s t-test; experiments were performed at least three times). c Confocal images of U2OS cells transfected with the indicated cDNAs encoding SSH-tagged variants of TEX264 and stained with an anti-HA antibody. TEX264ΔLRR image was deconvolved. Cells were treated with 10 µM EdU for 30 min before fixation. EdU was labelled using a Click-iT™ Alexa Fluor imaging kit. White arrowheads indicate examples of co-localisation. Scale bars, 5 μm (large panels) and 2 μm (magnified panels). See also Supplementary Fig. 7C. d Same as in c, except cells were co-transfected with FLAG-tagged SPRTN and co-stained with an anti-FLAG antibody. e Representative images of HeLa cells transfected with the indicated siRNA and stained with an antibody against γH2AX (phosphorylated on Ser139). f Quantification of the mean nuclear γH2AX intensity for experiment in e (****P < 0.0001; one-way ANOVA; red line indicates mean values). At least 100 nuclei were measured per condition. Scale bar, 20 μm. g, Immunoblots to confirm the efficacy of TEX264 and TOP1 depletions. h Proposed model: TEX264 is tethered at the nuclear periphery by its LRR. TEX264 binds to unmodified and SUMO1-modified TOP1 and counteracts TOP1cc accumulation by recruiting p97-SPRTN sub-complexes to TOP1ccs. S1 denotes SUMO1. Source data are available online.