Extended Data Fig. 2: KCTD10 negatively regulates transcription.
From: KCTD10 is a sensor for co-directional transcription–replication conflicts
a-b, nascent transcription assay in KCTD10-deficient or control U2OS cells pre-treated with PG490 (1 μM) or DMSO for 4 h prior to EU labeling. a, quantification of EU nuclear intensity. Data are presented as mean values ± SD (n = 3 biologically independent experiments). b, representative images depicting EU intensity with color scaled to the EU fluorescence intensity. c, western blot from KCTD10-deficient or control U2OS cells treated with PG490 (1 μM) or DMSO for 4 h. d-e, nascent transcription assay in KCTD10-deficient or control U2OS cells co-stained with fibrillarin. d, quantification of EU nuclear intensity. Data are presented as mean values ± SD (n = 3 biologically independent experiments). e, representative images and analysis strategy to quantify EU intensity in the nucleolus and nucleoplasm. Each dot represents the mean EU intensity per nucleus for >50 nuclei per independent replicate; n = 3. f, quantification of fork speed in KCTD10-deficient or control U2OS cells, treated with PARPi (olaparib, 10 μM) as indicated. Data are presented as mean values ± SD (n = 150 fibers examined over 3 independent experiments). g, schema depicting how PLA assays quantify TRCs. h, single antibody controls for PLAs in Fig. 1i,j. i, schema for head-on and co-directional TRC reporters8. j-m, DNA/RNA hybrid dot blot assay from KCTD10-deficient or control U2OS cells treated with ICRF193 (100 nM) or DMSO for 4 h. Purified DNA/RNA samples were treated with the indicated nucleases prior to blotting. j, representative blot. k, quantification of undigested blots. l, quantification of samples treated with RNase H. m, quantification of samples after treatment with RNase T1. Data are presented as mean values ± SD (n = 3 biologically independent experiments). Scalebars = 10 μm. p-values were calculated with a one-way ANOVA with Bonferroni’s multiple comparisons test.
