Fig. 1: KCTD10 reciprocally regulates DNA replication and transcription.
From: KCTD10 is a sensor for co-directional transcription–replication conflicts
a, γH2AX foci from KCTD10-deficient U2OS cells. Left, representative images. Right, quantification of foci. Data are mean ± s.d. (n = 3 biologically independent experiments). shKCTD10, shRNA targeting KCTD10; shScr, scramble shRNA. Scale bars, 10 μm. b, Western blot from KCTD10-deficient and control (Scr) U2OS cells. c, MTS assay in KCTD10-deficient or control U2OS cells with the TOP2 inhibitor ICRF193. d, Nascent DNA imaging in KCTD10-deficient or control U2OS cells pulsed with EdU for 30 min. Each dot represents the ratio of cells with EdU foci to cells with pan-nuclear EdU staining. Data are mean ± s.d. (n = 3 biologically independent experiments). Scale bars, 10 μm. e, DNA fibre assays in KCTD10-deficient or control U2OS cells pulsed with CldU and IdU as shown. Data are mean ± s.d. (n = 150 fibres examined over 3 independent experiments). f, Nascent transcription imaging in KCTD10-deficient or control U2OS cells pulsed with EU for 1 h. Data are mean ± s.d. (n = 3 biologically independent experiments). a.u., arbitrary units; max, maximum; min, minimum. Scale bars, 10 μm. g,h, DNA fibre assays in KCTD10-deficient or control U2OS cells, treated with the transcription inhibitor PG490 or PARPi (Extended Data Fig. 2f). g, Schema for fibre experiments (top) and representative fibre tracts (bottom). h, Quantification of fork speed in KCTD10-deficient or control U2OS cells, treated with PG490 (1 μM) as indicated. Data are mean ± s.d. (n = 150 fibres examined over 3 independent experiments). i,j, PLA for POLR2A–PCNA in KCTD10-deficient or control U2OS cells. i, Representative images. Scale bars, 10 μm. j, Quantification of foci. Data are mean ± s.d. (n = 200 cells examined over 3 independent experiments). P values were calculated with a two-tailed extra sum of squares F-test (c) or with one-way ANOVA with Bonferroni’s multiple comparisons test.
