Fig. 1
Dox induces nucleolar stress in hCmPCs in absence of ROS, apoptosis and cytotoxicity. a hCmPCs treated with 1 ÎĽM Dox for 4 h and 8 h (right upper and lower panels, respectively) or DMSO as control (w/o Dox; left upper and lower panels). Representative images of immunofluorescence of NPM (green) and nuclei counterstained with DAPI (blue). NPM is localised within nucleoli in control-treated cells and delocalises into the nucleoplasm upon Dox treatment. Scale bar 10ÎĽm. b Dox inhibits new rRNA synthesis in hCmPCs. Pre-rRNA 45S levels were measured by qRT-PCR and normalised to 18S expression (n = 4 for each group *P < 0.05; ***P < 0.001 vs Ctrl w/o Dox t=0h). c Representative image of DCF FACS measurements of hCmPCs treated or not with 1ÎĽM Dox for 8h. Serum starved hCmPCs were treated with 1 ÎĽM Dox for 8h. After treatment, cells were treated with DCF for ROS detection. d Bar graph representing mean fluorescence intensity (MFI) of DCF. Dox treatment does not significantly modulate ROS compared to untreated cells (n = 4 for each group). e Representative image of Annexin V FACS measurements of hCmPCs treated or not with 1ÎĽM Dox for 8, 24, 48, and 72h. After treatment, cells were stained with Annexin V for apoptosis detection. 30 min of 10mM H2O2 was used as positive control. Dox treatment significantly induced apoptosis at 48h and 72h compared Ctrl cells (n = 4 for each group; *P < 0.05 vs t=0h). f Representative image of cytotoxicity in Dox-treated hCmPCs. hCmPCs were treated with 1 ÎĽM Dox and analysed for cytotoxicity at different time points described in the figure. hCmPCs exhibited a significant cell toxicity only at 48h of treatment with Dox (n = 6; *P < 0.05)