Extended Data Fig. 7: ELOF1 KO impairs recruitment of UVSSA but not CSB.
From: Elongation factor ELOF1 drives transcription-coupled repair and prevents genome instability
a, Left panel: Schematic of the genomic locus of CSB and used strategy for generating the homozygous CSB-mScarletI-HA KI cell line. Half arrows indicate primer locations. Middle and right panel: Genotyping PCR and immunoblot for CSB-KI cell line. b, Left panel: Schematic of the genomic locus of UVSSA and used strategy for generating the homozygous UVSSA- mScarletI-HA KI cell line. Half arrows indicate primer locations. Middle and right panel: Genotyping PCR and immunoblot for UVSSA-KI cell line. Experiments depicted in a and b were performed two times with similar results. c, Left panel: CSB mobility was determined by FRAP analysis of CSB-mScarletI after the indicated treatments. THZ1: 1 hour treatment (2 µM) before UV-C irradiation (4 J/m2) or mock treatment. Right panel: Relative immobile fraction of CSB as determined by FRAP analysis. Plotted values represent mean ± SEM and are normalized to mock treated. NT n = 32; UV n = 28; THZ1 n = 15; THZ1+UV n = 18 cells analyzed across 2 independent experiments. d, Same as C but for UVSSA-mScarletI. NT n = 10; UV n = 16; THZ1 n = 16; THZ1+UV n = 17 cells analyzed across 2 independent experiments. e,f, FRAP analyses of CSB-mScarletI (e) or UVSSA-mScarletI (f) mobility after transfection with indicated siRNAs in individual graphs. Cells were mock treated (NT) or analyzed directly (UV) or 5 hours (5hr UV) after irradiation with 4 J/m2 UV-C. g, Relative fluorescence intensity of UVSSA in UVSSA-KI cells transfected with indicated siRNAs as determined by live-cell imaging. Plotted values represent mean ± SEM. siCTRL NT n = 30, UV+5h UV n = 21; siELOF1 #1 NT n = 38, UV n = 34, 5h UV n = 16; siELOF1 #2 NT+UV n = 19, 5h UV n = 16 cells analyzed across 4 independent experiments for siCTRL and 3 for siELOF1 and siCSB. h, FRAP analysis of CSB in CSB-KI cells transfected with indicated siRNAs 2 hours after UV. VCPi: VCP inhibitor (5 µM) was directly added after UV-C (4 J/m2). i, Immunoblot of chromatin fraction of indicated cell lines 1 hour after 12 J/m2 UV-C or mock treatment. NAEi = 1 hour treatment with NEDDylation inhibitor (10 µM). SSRP1 is shown as loading control. j, Immunoblot of chromatin fraction of HCT116 cells transfected with indicated siRNAs 1 hour after 12 J/m2 UV-C or mock treatment. SSRP1 is shown as loading control. Immunoblots depicted in i and j were executed two times with similar results. Numerical data and uncropped blots are provided in source data.
