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. 2021 Apr 19;10:e61983. doi: 10.7554/eLife.61983

Figure 2. Apoptotic ROCK1nc mouse embryo fibroblasts (MEFs) do not undergo forceful contraction.

(A) Homozygous ROCK1wt (upper panels) or ROCK1nc (lower panels) MEFs were serum-starved overnight, then treated with tumour necrosis factor α (TNFα) (50 ng/mL) plus cycloheximide (CHX) (10 µg/mL) to induce apoptosis. Differential interference contrast time-lapse microscopy images were acquired at 3 min intervals after the addition of TNFα plus CHX. Scale bar = 50 µm. Right panels: scanning electron microscope images. Scale bar = 10 µm. (B) Schematic diagram of a cell on elastic micropillars. (C) Homozygous ROCK1wt MEFs plated on elastic micropillars were serum-starved overnight, then treated with TNFα plus CHX to induce apoptosis. Brightfield time-lapse microscopy images were acquired at 2 s intervals after the commencement of cell contraction over 10 min. Right panel: force heat map indicating the magnitude of the force applied to each individual pillar. Scale bar = 10 µm. (D) Homozygous ROCK1wt (upper panels) or ROCK1nc (lower panels) MEFs were serum-starved overnight, then treated with TNFα plus CHX to induce apoptosis. Brightfield time-lapse microscopy images were acquired at 2 s intervals after the commencement of cell contraction over 10 min. Right panels: force vector map with direction and magnitude of the forces applied to each individual pillar. Scale bar = 20 nN. Mean maximum force is the average of the maximum forces applied to each pillar over 10 min. (E) Mean maximum force for untreated or TNFα plus CHX-treated homozygous ROCK1wt (green) or ROCK1nc (red) MEFs. Means ± SD n=27–50 individual cells. One-way ANOVA with post-hoc Tukey’s multiple comparison test; ns: not significant.

Figure 2.

Figure 2—figure supplement 1. Characterization of ROCK1wt and ROCK1nc mouse embryo fibroblasts (MEFs).

Figure 2—figure supplement 1.

(A) Cell numbers after plating 4 × 103 ROCK1wt and ROCK1nc MEFs at daily intervals up to 4 days. Means ± standard deviation, n = 5 independent replicates. (B) Example images used for high content analysis. F-actin staining with Oregon Green conjugated phalloidin, phosphorylated myosin light chain (MLC) (Ser19) staining with mouse primary antibody and AF594-conjugated (red) goat anti-mouse secondary antibody. Nuclei were stained with DAPI (blue). High content image analysis of ROCK1wt and ROCK1nc MEFs was performed using an Operetta high content imaging system as described in George et al., 2007 to determine (C) nuclear area, (D) nuclear roundness, (E) nuclear width, (F) nuclear length, (G) nuclear width to length ratio, (H) cell area, (I) cell roundness, (J) cell width, (K) cell length and (L) cell width to length ratio. Means ± SEM, n = 3 independent replicates.