Fig. 6: SPRTN-independent TC-DPC repair.
From: Transcription-coupled DNA–protein crosslink repair by CSB and CRL4CSA-mediated degradation
a, Quantification of recovery of transcription in RPE1 WT and CSB KO cells transfected with short interfering RNA (siRNA) targeting SPRTN (siSPRTN) or a control siRNA (siCtrl) after a 30 min pulse of 1 mM FA. After FA washout, cells were left to recover for the indicated times, including a 30 min pulse labelling with EU before fixation. RFI of EU were normalized to untreated levels and set to 100%. Black lines indicate the average integrated density ± s.e.m. from three independent experiments. n (left to right) = 777, 634, 742, 757, 509, 481, 491, 458, 494, 480, 448, 616, 333, 371, 356 and 349 cells. Unpaired two-tailed t-test. b, Relative immobile fraction of CSB FRAP in non-replicating RPE1 CSB–mScarlet-I cells transfected with the indicated siRNAs and treated with 1 μM palbociclib 24 h before FRAP. Cells were treated with 300 μM FA for 30 min and followed for the indicated times. Values represent the mean ± s.e.m. from three independent experiments. n (left to right) = 49, 51, 55, 46, 37, 41, 34, 39, 45, 55, 56, 55, 46, 60 and 34 cells. Unpaired two-tailed t-test. c, Relative survival of non-replicating RPE1 cells as determined by Alamar Blue staining. RPE1 cells, arrested in G1 by treatment with 1 μM palbociclib, were treated with the indicated doses of FA for 1 h and allowed to recover for 4 days. Metabolic activity was assayed as a measure of cell viability. Values represent the mean ± s.e.m. from four independent experiments and were normalized to untreated cells. Unpaired two-tailed t-test at 5 mM FA. d, GFP–Pol II FRAP in WT and CSB KO neurons. GFP–RPB1 KI iPS cells were differentiated into post-mitotic neurons through neurogenin-2 induction. FRAP in two different clones (A and B) was performed after treatment with 300 μM FA for 90 min. Graph represents values from eight cells. Source numerical data are available in the source data.
