Fig. 2.

Loss of ROCK1 expression impairs apoptotic membrane blebbing and ApoBD formation. a Loss of ROCK1 protein expression with CRISPR/Cas9-mediated ROCK1 gene disruption in three Jurkat T cell clonal populations (ROCK1^{−/− C1}, ROCK1^{−/− C2} and ROCK1^{−/− C3}) was validated by immunoblot analysis. Caspase 3/7 activity in ROCK1^−/− cells induced to undergo apoptosis by UV irradiation (b) or anti-Fas treatment (c) for 4 h (n = 3). d Time-lapse microscopy images monitoring the morphologies of ROCK1^−/− cells undergoing anti-Fas induced apoptosis. Arrow, cell of interest. Quantitation of apoptotic ROCK1^−/− cells that underwent surface blebbing (e) and dynamic blebbing (f), and total blebbing time (g) (n = 3). h Representative flow cytometry plots of apoptotic cells and ApoBDs generated by ROCK1^−/− cells treated with anti-Fas and trovafloxacin (40 µM, PANX1 inhibitor, to promote apoptopodia-dependent ApoBD formation). i Formation of ApoBDs from apoptotic ROCK1^−/− cells treated with anti-Fas and trovafloxacin, as measured by flow cytometry (n = 3). For quantitative analysis of blebbing time, all cells showing surface or dynamic blebbing was included. The duration between the first detection of blebbing to termination of blebbing was measured. ApoBD formation index determined by the number of ApoBDs divided by the number of A5⁺ apoptotic cells. a–i Cas9, Jurkat T cells expressing Cas9 only was used as a control. Error bars represent s.e.m. Data are representative of at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, NS = P > 0.05, unpaired Student’s two-tailed t-test