Fig. 5: Transcription stress triggered by OH-ME and role of CSA.
Assessment of de novo RNA synthesis upon OH-ME treatment for 24 h in HepG2 (A, B) and HeLa WT versus HeLa CSA–/– cells (C, D). Actinomycin D (ActD) was included as positive control. EU incorporation was analyzed by click chemistry with FAM-Azide (green), while nuclei were visualized by DAPI. Images were acquired by confocal microscopy and processed by Zen software. Representative images (A, C, scale bar: 10 µm) and the quantitative evaluation (B, D) are shown (n = 3–5). E, F Western blot detection of RPB1 and phospho-Ser2/Ser5 RPB1 (pRPB1) in HepG2 cells upon OH-ME treatment for 24 h in the absence or presence of the proteasome inhibitor MG132 (10 µM, added 6 h before cell harvest). In addition, HepG2 cells were exposed to UV-C (20 J/m2) followed by 2 h incubation with or without MG132. Representative western blots (E) and densitometric evaluation of RPB1 levels (F) are shown. Hsp90 served as loading control (n = 3). G–I Subcellular localization of RPB1 and pRPB1 in HepG2 cells 24 h after OH-ME treatment with or without proteasome inhibition by MG132 (10 µM, added 6 h prior to cell harvest). Cells were stained for RPB1 (red) and pRPB1 (green), while the nuclei were visualized by DAPI (blue). Images were acquired by confocal microscopy and processed by Zen software. Representative images (G, scale bar: 10 µm) and the quantitative evaluation of cytoplasmic RPB1 (H) and pRPB1 (I) levels are shown (n = 4). All data are given as mean + SEM. *p < 0.05, **p < 0.01, ****p < 0.0001.
