ROCK1-deficient mice display higher survivability and reduced ROS levels in response to stress. Kaplan Meier survival curve of WT and ROCK1^−/−mice after PHZ treatment. (A) Seventeen WT mice and 15 ROCK1^−/− mice treated with a single intraperitoneal injection of PHZ (90 mg/kg). Data shown is pooled from 3 independent experiments (*P < .05). (B) PB (20 μL) was washed and resuspended in prewarmed PBS and loaded with 10μM 5-(and 6-)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H₂DCFDA) in the dark for 30 minutes at 37°C. A representative histogram shows percentage of ROS probe-positive cells in PB of both genotypes. (C) Bar graph representing the mean ± SD of ROS levels in the peripheral blood of WT and ROCK1^−/− mice at 5 days after PHZ treatment (n = 3 mice for each genotype, *P < .05). (D) BM cells (5 × 10⁶) were immunostained for 30 minutes at 4°C with PE-conjugated anti-Ter119 antibody washed and resuspended in prewarmed PBS and loaded with 10μM CM-H₂DCFDA in the dark for 30 minutes at 37°C, 5% CO₂. Top right quadrant in each dot blot indicates percentage of ROS probe-positive erythroid cells in the BM of both genotypes. (E) Bar graph represents the mean ± SEM of ROS levels in erythroid cells in the BM of WT and ROCK1^−/− mice at 5 days after PHZ treatment (n = 3 per genotype, *P < .05). (F) Splenocytes (5 × 10⁶) were immunostained for 30 minutes at 4°C with PE-conjugated anti-Ter119 antibody washed and resuspended in prewarmed PBS and loaded with 10μM CM-H₂DCFDA in the dark for 30 minutes at 37°C, 5% CO₂. Top right quadrant in each dot blot indicates percentage of ROS probe-positive erythroid cells in spleen of both genotypes. (G) Bar graph represents the mean ± SEM of ROS levels in erythroid cells in the spleen of WT and ROCK1^−/− mice, 5 days after PHZ treatment. (n = 3 mice per genotype, *P < .05).