Loss of Arp2/3 complex affects volume regulation and osmotic stress signaling. (A) Graph showing cell death rate of NS and 2×KD cells in hyperosmotic conditions. (B, left) Representative images showing Arp2-EGFP–expressing cells under iso- and hyperosmotic conditions. (right) Quantification of peripheral Arp2 under iso- and hyperosmotic conditions. ****, P < 0.001 by Student’s t test. Arp2-EGFP signals were identified manually, and its length versus the whole cell perimeter was defined as the peripheral Arp2 ratio and plotted. (C) Graph showing cell volume of NS and 2×KD cells in hypo (50% mixture of H₂O and DMEM media)-, iso-, and hyper (0.25 M sorbitol-containing media)-osmotic conditions. ****, P < 0.001; **, P < 0.01 by Student’s t test. (D) Blot showing p-p38, p38, and GAPDH levels of NS and 2×KD cells under hyperosmotic conditions at different time points. (E) Blot showing p-p65, p65, p-p38, p38, and GAPDH levels of NS and 2×KD cells in hypoosmotic conditions at different time points. Fold changes in band intensity averaged across the three experiments (normalized to loading control and experimental control) are shown below each band. (F, i) Representative images showing endogenous p34Arc and F-actin (phalloidin) localization in the actin cortex. (ii) Representative images showing wild-type (WT) and 2×KD cells expressing Lifeact-EGFP. (iii) Fluorescent intensity profiles of the Lifeact signal near the cell edge were analyzed and plotted. Error bars show 95% CI. A.U., arbitrary unit.