Figure 1
Figure 1. Increased recruitment of macrophages and neutrophils into the inflamed peritoneum of ROCK1−/− mice. (A) Western blot analysis demonstrating the expression of ROCK1 and ROCK2 in BMMs from WT and ROCK1−/− mice. Equal amounts of cell lysate were probed with antibodies specific for the coiled-coil region of ROCK1 or ROCK2, respectively. The same blot was reprobed for β-actin to show protein loading in each lane. Lanes 1 and 2 represent lysates derived from WT and ROCK1−/− BMMs, respectively. The top arrow indicates expression of ROCK1 in WT BMMs but not in ROCK1−/− BMMs; the middle arrow indicates the expression of ROCK2 in both WT and ROCK1−/− BMMs; the bottom arrow indicates β-actin expression in WT and ROCK1−/− BMMs; n = 3. (B) WT and ROCK1−/− mice were given intraperitoneal injections of 4% thioglycollate. Peritoneal lavage was collected 4 days after injection, and mature macrophage-specific marker F4/80 expression was examined by flow cytometry. Top left quadrant of each dot blot represents the percentage of PE-conjugated F4/80-positive cells in WT and ROCK1−/− peritoneum after PBS or thioglycollate injections. Dot blots shown are representative of macrophages recruited into the peritoneal cavity of WT and ROCK1−/− mice, analyzed after PBS or thioglycollate injection. Similar results were obtained in 7 other mice from each genotype examined. (C) A representative bar graph shown here is the total number of macrophages recruited into the peritoneal cavity. Results shown are from one independent experiment containing 3 pairs of mice from each genotype after PBS or thioglycollate injection. WT vs ROCK1−/− mice, *P < .01. Similar results were obtained in 2 other independent experiments using 5 mice of each genotype (n = 3, 8 pairs of WT and ROCK1−/− mice). (D) Representative micrograph shown is a cytospin preparation of macrophages elicited by thioglycollate treatment from the peritoneal cavity of WT and ROCK1−/− mice. Peritoneal macrophages were stained with Wright-Giemsa. Images were acquired through a Zeiss Axioskop 2 Plus microscope equipped with a Plan-Neofluar 20×/0.5 objective lens, and were captured with an Axiocam MRC-5 camera and Axiovision 4 software (all from Carl Zeiss). (E) Increased recruitment of neutrophils to the inflamed peritoneum of ROCK1−/− mice. WT and ROCK1−/− mice were given intraperitoneal injections of 4% thioglycollate. Peritoneal lavage was collected after 4 hours of injection, and Gr-1/Mac-1 expression was examined by flow cytometry. Top right quadrant of each dot blot represents percentage of Gr-1 and Mac-1 double-positive cells in the peritoneum after PBS or thioglycollate injections. Dot blots shown are representative of neutrophils migrated into the peritoneum of WT and ROCK1−/− mice, analyzed after PBS or thioglycollate injection. Similar results were obtained in 4 other mice from each genotype examined. (F) A representative bar graph shows the total number of neutrophils recruited into the peritoneal cavity. Results are from one independent experiment containing 3 pairs of mice of each genotype after PBS or thioglycollate injection. WT vs ROCK1−/− mice, *P < .05. Similar results were obtained from one other independent experiment containing 2 mice of each genotype (n = 2, 5 pairs WT and ROCK1−/− mice).

Increased recruitment of macrophages and neutrophils into the inflamed peritoneum of ROCK1−/− mice. (A) Western blot analysis demonstrating the expression of ROCK1 and ROCK2 in BMMs from WT and ROCK1−/− mice. Equal amounts of cell lysate were probed with antibodies specific for the coiled-coil region of ROCK1 or ROCK2, respectively. The same blot was reprobed for β-actin to show protein loading in each lane. Lanes 1 and 2 represent lysates derived from WT and ROCK1−/− BMMs, respectively. The top arrow indicates expression of ROCK1 in WT BMMs but not in ROCK1−/− BMMs; the middle arrow indicates the expression of ROCK2 in both WT and ROCK1−/− BMMs; the bottom arrow indicates β-actin expression in WT and ROCK1−/− BMMs; n = 3. (B) WT and ROCK1−/− mice were given intraperitoneal injections of 4% thioglycollate. Peritoneal lavage was collected 4 days after injection, and mature macrophage-specific marker F4/80 expression was examined by flow cytometry. Top left quadrant of each dot blot represents the percentage of PE-conjugated F4/80-positive cells in WT and ROCK1−/− peritoneum after PBS or thioglycollate injections. Dot blots shown are representative of macrophages recruited into the peritoneal cavity of WT and ROCK1−/− mice, analyzed after PBS or thioglycollate injection. Similar results were obtained in 7 other mice from each genotype examined. (C) A representative bar graph shown here is the total number of macrophages recruited into the peritoneal cavity. Results shown are from one independent experiment containing 3 pairs of mice from each genotype after PBS or thioglycollate injection. WT vs ROCK1−/− mice, *P < .01. Similar results were obtained in 2 other independent experiments using 5 mice of each genotype (n = 3, 8 pairs of WT and ROCK1−/− mice). (D) Representative micrograph shown is a cytospin preparation of macrophages elicited by thioglycollate treatment from the peritoneal cavity of WT and ROCK1−/− mice. Peritoneal macrophages were stained with Wright-Giemsa. Images were acquired through a Zeiss Axioskop 2 Plus microscope equipped with a Plan-Neofluar 20×/0.5 objective lens, and were captured with an Axiocam MRC-5 camera and Axiovision 4 software (all from Carl Zeiss). (E) Increased recruitment of neutrophils to the inflamed peritoneum of ROCK1−/− mice. WT and ROCK1−/− mice were given intraperitoneal injections of 4% thioglycollate. Peritoneal lavage was collected after 4 hours of injection, and Gr-1/Mac-1 expression was examined by flow cytometry. Top right quadrant of each dot blot represents percentage of Gr-1 and Mac-1 double-positive cells in the peritoneum after PBS or thioglycollate injections. Dot blots shown are representative of neutrophils migrated into the peritoneum of WT and ROCK1−/− mice, analyzed after PBS or thioglycollate injection. Similar results were obtained in 4 other mice from each genotype examined. (F) A representative bar graph shows the total number of neutrophils recruited into the peritoneal cavity. Results are from one independent experiment containing 3 pairs of mice of each genotype after PBS or thioglycollate injection. WT vs ROCK1−/− mice, *P < .05. Similar results were obtained from one other independent experiment containing 2 mice of each genotype (n = 2, 5 pairs WT and ROCK1−/− mice).

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