Extended Data Fig. 8: TC-DPC repair is independent of SPRTN.
From: Transcription-coupled DNA–protein crosslink repair by CSB and CRL4CSA-mediated degradation
a: Immunoblot of RPE1 cells, transfected with siRNA against SPRTN, stained with antibodies against SPRTN and Tubulin. This experiment was performed twice with similar results. b: Representative pictures of transcription levels in RPE1 WT and CSB KO cells transfected with siRNAs against SPRTN. Quantification is shown in Fig. 6a. Scale bar, 50 = μm. c: Sequencing of UVSSA and SPRTN mutations in RPE1 cells. d: Immunoblot for the indicated proteins in various RPE1 KO cell lines. This experiment was performed twice with similar results. e: Clonogenic survival assay in RPE1 cells after varying doses of UV and FA. Graph represents the average ±S.E.M. from 3 independent experiments. Unpaired two-tailed t-test at 3 J m-2 UV and 2 mM FA. f: Representative pictures of transcription levels in WT, CSA KO, CSB KO, UVSSA KO and SPRTN −/+ RPE1 cells. Scale bar = 50 μm. g: Quantification of the transcription levels as shown in (f). Relative fluorescence intensities (RFI) of EU were normalized to mock-treated levels and set to 100%. Black lines indicate average integrated density ±S.E.M. n = 424, 407, 408, 654, 352, 308, 345, 345, 420, 385, 493, 413, 508, 482, 576, 626, 302, 283, 294, 304 cells (left to right) from 2 independent experiments. h: CSB-mScarlet-I FRAP with in cells non-replicating cells which were treated with 1 μM Palbociclib (CDKi) for 24 hr prior to FRAP after 1 mM FA for 30 min. Graph is an average of n = 35, 37, 40, 40, 40 cells (up to down) from 4 independent experiments. i: Relative immobile fractions of mScarlet-I-CSB FRAP as in (h). Values represent mean ± S.E.M. Unpaired two-tailed t-test. j: CSB-mScarlet-I FRAP with in cells transfected with control (Ctrl) siRNA. Cells were treated with 1 μM Palbociclib (CDKi) for 24 hr prior to FRAP after 1 mM FA for 30 min. Relative immobile fraction as shown in Fig. 6b. Graph is an average of n = 49, 46, 34, 55, 46 cells (up to down) from 3 independent experiments. k: CSB-mScarlet-I FRAP with in cells transfected with siCSA RNA. Cells were treated with 1 μM Palbociclib (CDKi) for 24 hr prior to FRAP after 1 mM FA for 30 min. Relative immobile fraction as shown in Fig. 6b. Graph is an average of n = 51, 37, 39, 56, 60 cells (up to down) from 4 independent experiments. l: CSB-mScarlet-I FRAP with in cells transfected with siSPRTN RNA. Cells were treated with 1 μM Palbociclib (CDKi) for 24 hr prior to FRAP after 1 mM FA for 30 min. Relative immobile fraction as shown in Fig. 6b. Graph is an average of n = 55, 41, 45, 55, 34 cells (up to down) from 3 independent experiments. m: Characterization of the CSB mutation in iPS GFP-RPB1 cells. This experiment was performed twice with similar results. Source numerical data and unprocessed blots are available in source data.
