Visualization and Quantification of DNA Damage and Autophagy

Cells on glass coverslips were treated with either MMV652103 or vehicle for 24 and 48 h and processed for immunofluorescence with antibodies against phospho H2A.X (#2577) (1:500) or LC3B (#2775) (1:200) (Cell Signaling Technology) as previously described (Bleloch et al., 2019[8 (link)]). Cells were imaged using confocal microscopy (Carl Zeiss LSM 880 with Fast Airyscan module confocal (Oberkochen, Germany)). Multiple z layers were acquired with 1 μM step width. Images were processed using ZEN 2012 imaging software (Carl Zeiss) and maximum intensity projections were generated. For quantification, mean fluorescence was measured from at least 20 fields of view per treatment condition and pooled from three independent replicates.