1. Autophagy mediates postslippage cell fate.
Autophagy mediates postslippage cell fate. A, Representative images of U2OS cells stably expressing shCtrl or shATG5 stained with SA-β-gal after 72-hour nocodazole (Noc) treatment. Plot shows percentage of SA-β-gal–positive cells. One-hundred cells per condition and data are mean ± SD of three independent experiments. Scale bar, 10 μm. B, U2OS cells were transfected with siCtrl or siATG5 followed by nocodazole treatment for 72 hours. Proliferation was assessed by the BrdU incorporation assay. Plot shows percentage of BrdU-positive multinucleated cells. One-hundred cells per condition and data are mean ± SD of three independent experiments. Scale bar, 20 μm. C, IB shows efficiency of ATG5 knockdown in U2OS cells transfected with siCtrl or siATG5 followed by nocodazole treatment for 72 hours. D, U2OS shCtrl or shATG5 cells carrying H2B-GFP were treated with nocodazole for 48 hours. Postslippage multinucleated cells were tracked individually by time-lapse microscopy for another 2 days. Red, cells that die postslippage. Black, cells that arrest postslippage. Fifty cells per condition and data are mean ± SD of two independent experiments. E, U2OS or RPE-1 cells were treated with nocodazole or paclitaxel in absence or presence of Baf A1 for 72 hours. Cell viability was assessed by CellTiter 96 One Solution Proliferation assay. F, Experimental scheme of clonogenic cell survival assay. Left, representative images of U2OS cells treated with the indicated drugs followed by crystal violet staining. Right, plot shows number of colonies counted using GelCount software. Eight wells per condition of 2 independent experiments. G, U2OS shCtrl or shATG5 cells were treated with indicated drugs for 72 hours and subjected to IB analysis. H, U2OS shCtrl or shATG5 cells were treated with nocodazole for 72 hours and subjected to IF. Plot shows percentage of γH2AX-positive multinucleated cells. One-hundred cells per condition and data are mean ± SD of two independent experiments. Scale bar, 20 μm. I, U2OS shCtrl or shATG5 cells were treated with nocodazole for 72 hours and subjected to IB analysis (*, P < 0.05; **, P < 0.005; ***, P < 0.001; ****, P < ; ns, not significant by Student t test).
Rekha Jakhar et al. Mol Cancer Res 2018;16:
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