Extended Data Fig. 7: KCTD10 recruits CUL3 to co-directional TRCs.
From: KCTD10 is a sensor for co-directional transcription–replication conflicts
a, co-immunoprecipitation assay for KCTD10 from the indicated fractions shown in Fig. 4a. b-c, PLA for POLR2A and Ubiquitin (Ub) in KCTD10-deficient U2OS cells treated with ICRF193 (100 nM) or DMSO for 4 h. b, representative images. c, quantification of foci. Data are presented as mean values ± SD (n = 200 cells examined over 3 independent experiments). d, representative images from PLA control for the indicated antibodies used throughout the manuscript. e-f, PLA for KCTD10-POLR2A in HEK293T cells expressing the codirectional-ECFP reporter (Extended Data Fig. 2i) treated with doxycycline (1 mg/mL) for 20 h and ICRF193 (100 nM) or DMSO for 4 h. e, quantification of foci. Data are presented as mean values ± SD (n = 150 cells examined over 3 independent experiments). f, representative images. g-h, PLA for POLR2A-PCNA in U2OS cells treated with ICRF193 (100 nM) and MLN4924 (1 μM) for 4 h. g, quantification of foci. Data are presented as mean values ± SD (n = 200 cells examined over 3 independent experiments). h, representative images. i-j, PLA for CUL3-POLR2A in U2OS cells treated with ICRF193 (100 nM) or etoposide (ETP, 1 μM) for 4 h. i, quantification of foci. Data are presented as mean values ± SD (n = 200 cells examined over 3 independent experiments). j, representative images. Error bars indicate the mean ± SD. Scalebars = 10 μm. p-values were calculated with a one-way ANOVA with Bonferroni’s multiple comparisons test.
