Extended Data Fig. 6: INTS12-mediated removal of lesion-stalled Pol II following UV irradiation.
From: Integrator subunit INTS12 links ribotoxic stress to transcription-coupled nucleotide excision repair
a, Soluble and chromatin fractions from untreated or UV-irradiated control and INTS12 KO cells were analyzed by WB. b, Control or CSA KO cells expressing INTS12-Flag or not (-) were either untreated or treated with UV irradiation (20 J/m2, 1 h recovery). Chromatin fractions (Input) and anti-Flag immunoprecipitates (IPs) were analyzed by WB. c, Lysates from parental or Halo-RPB1 U2OS cells were analyzed by WB. d, FRAP analysis of Halo-RPB1 U2OS cells that were either untreated or treated with UV (8 J/m2, 1 h recovery) or FA (0.3 mM, 1 h). The relative fluorescence intensity (RFI) of Halo-RPB1 was measured every second for 220 s and normalized to pre-bleach fluorescence intensity (set to 1.0). Right: relative immobile fractions of Halo-RPB1, which were calculated by comparing RFI after UV treatment to untreated samples. Data are the mean ± s.e.m. of n = 4 biologically independent samples for the untreated and FA-treated groups, and n = 6 for the UV-treated group. e, Chromatin fractions from control, CSB KO, or INTS12 KO cells treated with THZ and/or UV as indicated were analyzed by WB. f, Control or INTS12 KO cells were either untreated or treated with UV (20 J/m2) followed by CHX (100 µg/mL) treatment. Lysates from cells that were harvested at the indicated time points were analyzed by WB. g, Metaplots of PADD-seq signals around TSSs and TESs for active genes longer than 50 kb under the indicated conditions. Right: quantification of Pol II retention on damage sites by relative change of PADD-seq signals from 0.5 h to 2 h on active genes longer than 20 kb. P < 9.8E-159. h, R-loop levels in mock- or 4-NQO-treated control, INTS12 or CSB KO cells were detected by GFP-dRNH1. Bottom: Quantification of mean nuclear GFP-dRNH1 intensity per cell and are representative of three independent experiment (n = 231, 242, 202, 202, 241, 231). Red lines indicate the mean intensity in each group. Scale bar, 50 μm. Statistical analysis was performed using two-tailed unpaired t-tests. All Western blots are representative of three independent experiments.
