Enhanced stress erythropoiesis in ROCK1-deficient mice. WT and ROCK1^−/− mice were treated with PHZ (90 mg/kg at day 0). At the indicated time points, tissues were harvested. (A) Photomicrographs of spleens in the 2 genotypes are shown after PBS and PHZ treatment. (B) Line chart represents mean ± SEM of splenic weight of WT and ROCK1^−/− mice after PHZ injection at indicated time points (n = 6 mice per genotype for the 3 day time point; n = 3 mice per genotype for 5 day and PBS time point, *P < .05). (C) Splenocytes from WT and ROCK1^−/− mice were analyzed for the expression of erythroid cell surface marker Ter119 and CD71 in combination with forward scatter (FSC) parameter by flow cytometry after PHZ treatment. A representative left dot blot shows the Ter119^high-positive erythroblasts in the spleen at indicated each time points. Ter119^high-positive erythroblasts were further analyzed for CD71 expression and FSC parameter as seen in right contour plots at each time point. Percentages in top-right quadrant contour represents CD71^high, Ter119^high, and FSC^high; percentages in top-left quadrant represents CD71^high, Ter119^high, and FSC^low; percentages in bottom-left quadrant represents CD71^low, Ter119^high, and FSC^low erythroblast in spleen. (D) Line chart representing the mean ± SEM of CD71^high,Ter119^high, and FSC^high erythroid subset in the spleen of WT and ROCK1^−/− mice after PHZ injection at the indicated time points. For each analysis (n = 3 mice per genotype for the 3 day time point; n = 3 mice per genotype for the 5 day and PBS time point, *P < .05). (E) Line graph representing the mean ± SEM of CD71^high, Ter119^high, and FSC^high erythroid subset in the spleen of WT and ROCK1^−/− mice after PHZ injection at the indicated time points. For each analysis (n = 3 mice per genotype for the 3 day time point; n = 3 mice per genotype for the 5 day and PBS time point, *P < .05). (F) Histopathologic analysis of WT and ROCK1^−/− spleens after PHZ treatment. Spleens were harvested at indicated time points, fixed in 10% buffered formalin, sectioned, and stained with hematoxylin and eosin. Shown are representative spleen sections from WT and ROCK1^−/− mice after PHZ treatment. Arrows in left panel indicate clearly defined architecture of white pulp and red pulp in WT spleen 3 and 5 days after PHZ treatment. Arrows in right panel indicate enhanced extramedullary erythropoiesis in ROCK1^−/− spleens after PHZ treatment (n = 3 mice per genotype for each time point). (G) Splenocytes (5.0 × 10⁴) from WT and ROCK1^−/− mice were plated in a methylcellulose colony-forming assay in the presence of the indicated concentration of SCF and EPO. Colonies were enumerated on day 2 (for CFU-E) and on day 10 (for BFU-E). Bar graph represents the mean ± SD of colonies from 3 independent mice for each genotype 3 days after PHZ treatment plated in triplicates (*P < .05, ROCK^−/− vs WT). (H) Bar graph indicates the mean ± SD number of colonies from WT and ROCK1^−/− splenocytes on day 5 after PHZ treatment., ROCK^−/− versus WT (*P < .05, n = 3 mice for each genotype). (I) Histopathologic analysis of WT and ROCK1^−/− BM 5 days after PHZ treatment. Bones were harvested at indicated time points, fixed in 10% buffered formalin, sectioned, and stained with hematoxylin and eosin. Shown are representative BM sections from WT and ROCK1^−/− mice after PHZ treatment. Right panel arrows demonstrate hypercellular BM and increased red cell generation of ROCK1^−/− BM compared with WT BM after PHZ treatment (n = 3 mice per genotype for each time point). (J) Splenocytes from WT and ROCK1^−/− mice after were plated in a methylcellulose colony-forming assay in the presence of the indicated concentration of SCF, EPO, and BMP-4. Colonies were enumerated on day 5 for stress BFU-E. Bar graph represents the mean number ± SEM of colonies from 3 independent mice for each genotype plated in triplicates on day 5 after PHZ treatment (n = 3, *P < .05; ROCK^−/− vs WT). (K) Total RNA was isolated using the RNeasy Minikit (QIAGEN) and single-stranded cDNA was synthesized using AccuScript High Fidelity 1st Strand cDNA Synthesis Kit (Aligent Technologies) according to the manufacturer's instructions. Q-PCR was performed using an ABI PRISM 7900HT Sequence Detection System (Applied Biosystems) using the SYBR Green Master Mix (Applied Biosystems). For data analysis, ΔCT values were calculated using the expression of the ribosomal housekeeping gene L27 as an internal control. Relative expression of Bmp4 of was normalized with WT splenocytes treated with PHZ. Bar graph represents expression of BMP-4 in ROCK1^−/− splenocytes after PHZ treatment on day 5 (n = 3, *P < .05; ROCK^−/− vs WT).