Figure 6.

TDP1 promotes transcription recovery following alkylation-induced DNA damage. (a) Control MRC5 cells ‘WT’ grown on coverslips were maintained for 2 days in serum-free media, treated with DMSO ‘Mock’ or with 20 μM CPT ‘CPT’ for 1 h and either harvested immediately after treatment or incubated in CPT-free media for a subsequent 3 h to allow for transcription recovery. Cells were incubated with 0.1 mM 5-ethynl uridine (EU) for 30 min to label newly synthesized RNA, which was visualized by using the Click iT reaction with Alexa Flour azide 488. EU-labeled RNA was subjected to immunofluorescence analyses and DNA counterstained with 4',6-diamidino-2-phenylindole (DAPI). Representative micrographs are depicted. (b) TDP1^KD or Top1^KD MRC5 cells were examined for nascent RNA synthesis as described in (a). (c) Average fluorescence signal (arbitrary units ‘AU’) from 200 to 300 cells as described in (a) and (b) were quantified from three biological replicates ± s.e.m. (d) The indicated MRC5 cells were grown in serum-free medium and treated with 2 μg/ml MMS for 15 min at 37°C. Cells were either harvested immediately after treatment ‘MMS’ or incubated in MMS-free media for a subsequent 1 h ‘R’. Newly synthesized RNA was quantified and average fluorescence signal of EU-labeled RNA was quantified as described earlier from three biological replicates.