Fig. 6: Regulation of CSB in response to OH-ME triggered transcription-blocking lesions and recovery of transcription.
A FRAP analysis of CSB-mClover in HCT116-WT, CSA–/– and XPA–/– cells exposed to 25 µM OH-ME for 16 h. The percentage of CSB-mClover immobile fraction was determined by FRAP analysis (n = 3). B Endogenous CSB-mClover fluorescence in HCT116 CSB-mClover WT and UVSSA–/–. Cells were mock-treated or exposed to 25 μM OH-ME with or without 2 μM MG132 for 16 h, or UV-C irradiated (4 J/m2) followed by 16 h recovery prior to fixation and DAPI staining. Fluorescence was background-corrected using non-fluorescent HCT116 WT cells and normalized to non-irradiated condition (n = 3). C, D Evaluation of the de novo RNA synthesis after OH-ME treatment and recovery time in HepG2. Cells were incubated with 75 µM OH-ME for 16 h. After a recovery period of 0-24 h the cells were fixed, processed and the EU incorporation (green) analyzed as described. Images were captured by confocal microscopy and processed by Zen software. Representative images (C, scale bar: 10 µm) and the quantitative evaluation of EU incorporation (D) are shown (n = 5). E Analysis of soluble and chromatin-bound (p)RPB1 in HepG2 cells. Cells were treated with 75 µM OH-ME for 16 h and then harvested after 0-24 h of recovery time, followed by cell fractionation. The cytosolic marker Hsp90 and the chromatin marker Histone H3 served as loading controls. Representative western blot images are shown (n = 5). All data are given as mean + SEM, except for panel B (mean ± SD). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
