Fig. 3: KCTD10 interacts with PCNA and the RNAP complex through its PIP box.
From: KCTD10 is a sensor for co-directional transcription–replication conflicts
a, iPOND assay in U2OS cells treated with EdU for 15 min and DMSO or 1 μM ICRF193 for 4 h prior to EdU. b, PLA for KCTD10–PCNA in U2OS cells treated with 100 nM ICRF193 or DMSO for 4 h. Left, representative images. Right, quantification of foci. Data are mean ± s.d. (n = 200 cells examined over 3 independent experiments). Scale bars, 10 μm. c, Co-immunoprecipitation (IP) assay in HEK293T cells transiently expressing Flag–KCTD10 and treated with 1 μM ICRF193 or DMSO for 4 h. d,e, PLA for KCTD10–POLR2A in U2OS cells treated with indicated drugs for 4 h. d, Quantification of foci. Data are mean ± s.d. (n = 200 cells examined over 3 independent experiments). e, Representative images. PARPi used at 10 μM; PlaB, pladienolide B used at 200 nM; HU, hydroxyurea used at 250 μM; Aph, aphidicolin used at 4 μM. Scale bars, 10 μm. f, schema of KCTD10 truncations. IDR1 and IDR2, intrinsically disordered regions 1 and 2. g, Co-immunoprecipitation assay in HEK293T cells expressing the indicated constructs as depicted in f. EV, empty vector control. h, Co-immunoprecipitation assay in HEK293T cells co-expressing Flag–KCTD10 with the indicated point mutations and Halo–KCTD10 and treated with 1 μM ICRF193 for 4 h. WT, wild type. i, Quantification of PLA foci for POLR2A–PCNA in KCTD10-deficient and control cells rescued with wild-type KCTD10 or KCTD10(R167A) and treated with ICRF193 (100 nM) or DMSO for 4 h. Data are mean ± s.d. (n = 200 cells examined over 3 independent experiments). j, Schema depicting how KCTD10 might bind to PCNA and RNAPII. P values were calculated with a two-tailed unpaired t-test (b) or one-way ANOVA with Bonferroni’s multiple comparisons test (d,i).
