Journal: PLoS ONE

Article Title: Rho Kinases Regulate the Renewal and Neural Differentiation of Embryonic Stem Cells in a Cell Plating Density–Dependent Manner

doi: 10.1371/journal.pone.0009187

Figure Lengend Snippet: A. CCE cells seeded at 10 4 cells/cm 2 for 24 h were treated with Y-27632 (10 µM) or RA (10 µM) for 48 h. ROCK-1 and ROCK-2 proteins were analyzed by Western blotting. B. ROCK activities in CCE cells treated with Y-27632 or H-89 (10 µM) for 2 h. Each bar denotes mean ± SEM of three experiments.

Article Snippet: Antibodies for identifying murine CCE proteins included (1) monoclonal antibodies against Flk-1 and Oct3/4, goat polyclonal antibodies against nestin, GATA-4 and actin and rabbit polyclonal antibodies against NG2 (all from Santa Cruz Biotechnology); (2) rabbit polyclonal antibodies against ROCK-1 and monoclonal antibodies against βIII-tubulin from Chemicon (Temecula, CA); (3) rabbit polyclonal antibodies against SOX-1 and monoclonal antibodies against MAP2c from Abcam (Cambridge, UK) and (4) rabbit polyclonal antibodies against ROCK-2 from Upstate.

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Rho Kinases Regulate the Renewal and Neural Differentiation of Embryonic Stem Cells in a Cell Plating Density–Dependent Manner

doi: 10.1371/journal.pone.0009187

Figure Lengend Snippet: A . CCE cells transfected with selective ROCK-1 and ROCK-2 siRNA or control RNA were seeded at 10 4 cells/cm 2 for 48 h, and ROCK-1 and ROCK-2 proteins were analyzed by Western blotting. The blots were quantitated by densitometry. Each bar denotes mean ± SEM (n = 3). B. Colony formation and growing pattern were examined under phase-contrast microscopy. C . Cells at 1×10 4 cells/cm 2 were treated with ROCK-1 plus ROCK-2 siRNA vs. individual ROCK siRNA. Oct3/4 proteins were analyzed by Western blotting. D. Oct3/4 and SOX-2 proteins were analyzed in siRNA-treated cells seeded at 1×10 4 cells/cm 2 (left panel) vs. 2×10 3 cells/cm 2 (right panel). The blots were analyzed by densitometry. Each bar denotes mean ± SEM (n = 3).

Article Snippet: Antibodies for identifying murine CCE proteins included (1) monoclonal antibodies against Flk-1 and Oct3/4, goat polyclonal antibodies against nestin, GATA-4 and actin and rabbit polyclonal antibodies against NG2 (all from Santa Cruz Biotechnology); (2) rabbit polyclonal antibodies against ROCK-1 and monoclonal antibodies against βIII-tubulin from Chemicon (Temecula, CA); (3) rabbit polyclonal antibodies against SOX-1 and monoclonal antibodies against MAP2c from Abcam (Cambridge, UK) and (4) rabbit polyclonal antibodies against ROCK-2 from Upstate.

Techniques: Transfection, Western Blot, Microscopy

Journal: Journal of Clinical Investigation

Article Title: Rho-kinase/AMPK axis regulates hepatic lipogenesis during overnutrition

doi: 10.1172/jci63562

Figure Lengend Snippet: Figure 1. ROCK1 expression and activation increase in the liver of animal models with obesity and in humans with fatty liver disease. (A) Hepatic ROCK1 expression in mice fed a normal chow diet or an HFD at 18 weeks of age (n = 5 per group). (B–D) Hepatic ROCK1 activity in HFD-fed mice (18 weeks of age, n = 5 per group) (B), ob/ob mice (10 weeks of age, n = 5 per group) (C), and db/db mice (10 weeks of age, n = 5 per group) (D). Mice were fed either a normal chow diet or an HFD for 12 weeks from 6 weeks of age. Liver lysates (30 μg) were separated by SDS-PAGE. ROCK1 was visualized by immunoblotting and quantitated by densitometry. ROCK1 activity in liver lysates (300 μg) was measured by immune complex assay. (E) Hepatic ROCK1 expression in humans with or without fatty liver disease (n = 9−10 per group). (F) Relationship between hepatic ROCK1 levels and BMI, serum triglyceride, alanine transaminase (ALT), and aspartate transaminase (AST) levels in humans with or without fatty liver disease. Relationships were statistically analyzed by Pearson correla- tion coefficient. (G) Oil Red O–stained liver sections in humans with or without fatty liver disease. Scale bars: 100 μm. Values are means ± SEM. **P < 0.01 vs. chow, lean, or control (non–fatty liver human) by unpaired Student’s t test.

Article Snippet: The membranes were incubated with polyclonal antibodies against ROCK1 (H-85, sc-5560, Santa Cruz Biotechnology), ROCK2 (H-85, sc-5561, Santa Cruz Biotechnology), α2AMPK (Abcam), and actin (C-11, sc-1615, Santa Cruz Biotechnology).

Techniques: Expressing, Activation Assay, Activity Assay, SDS Page, Western Blot, Staining, Control

Journal: Journal of Clinical Investigation

Article Title: Rho-kinase/AMPK axis regulates hepatic lipogenesis during overnutrition

doi: 10.1172/jci63562

Figure Lengend Snippet: Figure 2. Hepatic ROCK1 deficiency protects from diet-induced obesity and insulin resistance and increases energy expenditure. Body weight (n = 14−16 per group) (A), body mass measured by an MRI (14 weeks of age, n = 6−10 per group) (B), fat mass (26 weeks of age, n = 4−6 per group) (C), daily food intake (n = 7 per group) (D), O2 consumption (n = 5−6 per group) (E), locomotor activity (n = 5−6 per group) (F), thermogenic gene expression in brown adipose tissue (BAT) and epididymal white adipose tissue (WAT) (n = 6−12 per group) (G), blood glucose during insulin tolerance test (ITT) (H) and glucose tolerance test (GTT) (I), serum insulin (J), random blood glucose (K), and serum leptin levels (L) were measured in ROCK1loxP/loxP (control) and albumin-Cre;ROCK1loxP/loxP (L-ROCK1–/–) mice fed an HFD (n = 6−10 per group for H–L). Mice were fed an HFD from 6 weeks of age. Epi, epididymal fat; Peri, perirenal fat; Mes, mes- enteric fat; AAC, area above the curve; AUC, area under the curve. O2 consumption and locomotor activity were assessed by CLAMS at 18 weeks of age. Thermogenic gene expression was measured from overnight-fasted mice at 22 weeks of age. ITT and GTT were performed at 16−17 weeks of age. Serum parameters were measured from overnight-fasted mice at 18 weeks of age. Values are means ± SEM. *P < 0.05 vs. control, **P < 0.01 vs. control by unpaired Student’s t test.

Article Snippet: The membranes were incubated with polyclonal antibodies against ROCK1 (H-85, sc-5560, Santa Cruz Biotechnology), ROCK2 (H-85, sc-5561, Santa Cruz Biotechnology), α2AMPK (Abcam), and actin (C-11, sc-1615, Santa Cruz Biotechnology).

Techniques: Activity Assay, Gene Expression, Control

Journal: Journal of Clinical Investigation

Article Title: Rho-kinase/AMPK axis regulates hepatic lipogenesis during overnutrition

doi: 10.1172/jci63562

Figure Lengend Snippet: Figure 3. Loss of ROCK1 decreases hepatic lipid accumulation by reducing de novo lipogenesis. (A–E) Liver weight (A), liver triglycerides (B), liver choles- terol (C), H&E-stained liver sections (D), and in vivo fractional rate of triglycerides and glycerol in the liver (E) were measured in ROCK1loxP/loxP (control) and albumin-Cre;ROCK1loxP/loxP (L-ROCK1–/–) mice fed an HFD (n = 5−10 per group). Mice were fed an HFD from 6 weeks of age. Liver weight, liver triglycerides, and liver cholesterol were measured at 26 weeks of age. In vivo fractional rate of triglycerides and glycerol was measured from body weight–matched mice at 12 weeks of age. Scale bars: 100 μm. FSR, fractional synthesis rate. (F) In vivo fractional rate of triglycerides and glycerol in the liver were measured in control and L-ROCK1–/– mice fed a normal chow diet at 10–11 weeks of age (n = 4−5 per group). (G) Liver triglyceride and cholesterol content was measured in control and L-ROCK1–/– mice fed a normal chow diet at 18 weeks of age (n = 8−12 per group). (H and I) Fatty acid uptake (H) and fatty acid oxidation (I) were measured in isolated primary hepatocytes of body weight–matched control and L-ROCK1–/– mice fed an HFD at 12 weeks of age (n = 6 per group). (J) Serum triglyceride levels were measured after injection of poloxamer 407 solution in body weight–matched control and L-ROCK1–/– mice fed an HFD at 12 weeks of age (n = 7 per group). (K) Gene expression of key molecules involved in lipogenesis, gluconeogenesis, glycolysis, fatty acid oxidation, and uptake in the liver was measured by quantitative PCR in control and L-ROCK1–/– mice fed an HFD at 26 weeks of age (n = 5−8 per group). Values are means ± SEM. *P < 0.05 vs. control, **P < 0.01 vs. control by unpaired Student’s t test.

Article Snippet: The membranes were incubated with polyclonal antibodies against ROCK1 (H-85, sc-5560, Santa Cruz Biotechnology), ROCK2 (H-85, sc-5561, Santa Cruz Biotechnology), α2AMPK (Abcam), and actin (C-11, sc-1615, Santa Cruz Biotechnology).

Techniques: Staining, In Vivo, Control, Isolation, Injection, Gene Expression, Real-time Polymerase Chain Reaction

Journal: Journal of Clinical Investigation

Article Title: Rho-kinase/AMPK axis regulates hepatic lipogenesis during overnutrition

doi: 10.1172/jci63562

Figure Lengend Snippet: Figure 4. Hepatic ROCK1 activation accelerates adiposity, insulin resistance, and hepatic lipid accumulation. Body weight (n = 11−12 per group) (A), blood glucose during ITT (14 weeks of age, n = 6−10 per group) (B), liver triglycerides (C), liver cholesterol (D), H&E-stained liver sections (n = 3 per group) (E), random blood glucose (16 weeks of age) (F), serum insulin (G), serum triglycerides (H), serum cholesterol (I), thermogenic gene expression in BAT and epididymal WAT (n = 5–7 per group) (J), and gene expression of key molecules involved in lipogenesis, gluconeogenesis, glycolysis, fatty acid oxidation, and uptake (n = 5–7 per group) (K) were measured in CA-ROCK1 (control) and albumin-Cre;CA-ROCK1 (L-CA-ROCK1) mice fed an HFD. Mice were fed an HFD from 6 weeks of age. Serum and hepatic parameters were measured from overnight-fasted mice at 18 weeks of age (n = 9−10 for group). Thermogenic gene expression was measured from overnight-fasted mice at 18 weeks of age. Scale bars: 100 μm. Values are means ± SEM. *P < 0.05 vs. control, **P < 0.01 vs. control by unpaired Student’s t test.

Article Snippet: The membranes were incubated with polyclonal antibodies against ROCK1 (H-85, sc-5560, Santa Cruz Biotechnology), ROCK2 (H-85, sc-5561, Santa Cruz Biotechnology), α2AMPK (Abcam), and actin (C-11, sc-1615, Santa Cruz Biotechnology).

Techniques: Activation Assay, Staining, Gene Expression, Control

Journal: Journal of Clinical Investigation

Article Title: Rho-kinase/AMPK axis regulates hepatic lipogenesis during overnutrition

doi: 10.1172/jci63562

Figure Lengend Snippet: Figure 5. Deleting hepatic ROCK1 inhibits development of hepatic steatosis in ob/ob mice. Blood glucose (n = 8−10 per group) (A), serum insulin (n = 6−11 per group) (B), glucose × insulin (n = 6−11 per group) (C), liver triglycerides (n = 6−13 per group) (D), liver cholesterol (n = 6−13 per group) (E), the image of H&E-stained liver sections (n = 3 per group) (F), gene expression of key molecules in lipogenesis, gluconeogenesis, glycolysis, fatty acid oxidation, and uptake (n = 5−10 per group) (G), body weight (H), serum triglyceride (I), and serum cholesterol (n = 9−13 per group) (J) in ROCK1loxP/loxP (control), liver-specific ROCK1-deficient (L-ROCK1–/–), ob/ob, and L-ROCK1–/–:ob/ob mice at 14 weeks of age. Blood glucose levels were measured from random-fed mice. Serum and hepatic parameters were measured from overnight-fasted mice. Scale bars: 100 μm. Values are means ± SEM. *P < 0.05, **P < 0.01 vs. control, #P < 0.01 vs. ob/ob by ANOVA with Fisher’s PLSD (protected least significant difference).

Article Snippet: The membranes were incubated with polyclonal antibodies against ROCK1 (H-85, sc-5560, Santa Cruz Biotechnology), ROCK2 (H-85, sc-5561, Santa Cruz Biotechnology), α2AMPK (Abcam), and actin (C-11, sc-1615, Santa Cruz Biotechnology).

Techniques: Staining, Gene Expression, Control

Journal: Journal of Clinical Investigation

Article Title: Rho-kinase/AMPK axis regulates hepatic lipogenesis during overnutrition

doi: 10.1172/jci63562

Figure Lengend Snippet: Figure 6. Endocannabinoid-induced lipogenesis is mediated via a ROCK1→AMPK signaling axis. (A) Endocannabinoid-induced ROCK1 activity was measured in the liver of C57BL/6 mice at 10 weeks of age and in HepG2 cells (n = 5−6 per group). (B) 2-AG–stimulated hepatic AMPK activity was measured in overnight-fasted ROCK1loxP/loxP (control) and albumin-Cre;ROCK1loxP/loxP (L-ROCK1–/–) mice fed a normal chow diet at 10 weeks of age (n = 6−9 per group). (C) Hepatic AMPK activity was measured in overnight-fasted L-ROCK1–/– and L-CA-ROCK1 mice fed an HFD at 18 weeks of age. ROCK1 and AMPK activity were measured by immune complex assay (n = 5−11 per group). (D) 2-AG–stimulated lipogenic gene expression was measured in control and L-ROCK1–/– mice fed a normal chow diet at 10 weeks of age. Mice were injected i.p. with 2-AG and sacrificed 4 hours later (n = 4−6 per group). (E and F) 2-AG–induced lipogenic gene expression (E) and 2-AG–induced lipogenic rate (F) were measured in isolated primary hepatocytes from control and L-ROCK1–/– mice fed a normal chow diet at 8 weeks of age (n = 4−10 per group). Isolated primary hepatocytes were treated with 2-AG for 6 hours and har- vested for mRNA extraction. Gene expression was measured by quantitative PCR. Values are means ± SEM. *P < 0.05, **P < 0.01 vs. vehicle, †P < 0.05,

Article Snippet: The membranes were incubated with polyclonal antibodies against ROCK1 (H-85, sc-5560, Santa Cruz Biotechnology), ROCK2 (H-85, sc-5561, Santa Cruz Biotechnology), α2AMPK (Abcam), and actin (C-11, sc-1615, Santa Cruz Biotechnology).

Techniques: Activity Assay, Control, Gene Expression, Injection, Isolation, Extraction, Real-time Polymerase Chain Reaction

Journal: Journal of Clinical Investigation

Article Title: Rho-kinase/AMPK axis regulates hepatic lipogenesis during overnutrition

doi: 10.1172/jci63562

Figure Lengend Snippet: Figure 7. Metformin suppresses lipogenesis by inhibiting ROCK1. (A–G) Blood glucose (n = 7−8 per group) (A), serum insulin (n = 7−10 per group) (B), hepatic AMPK activity (n = 5−10 per group) (C), hepatic ROCK1 activity (n = 5−10 per group) (D), liver triglyceride (n = 4−9 per group) (E), liver cholesterol (n = 4−9 per group) (F), and lipogenic gene expression (n = 5−10 per group) (G) were measured in mice fed an HFD treated with metformin. HFD-fed mice (from 6 weeks of age) were treated with metformin for 12 weeks from 22 weeks of age. (H–J) Metformin-stimulated lipogenic rate (H), ROCK1 activity (I), and AMPK activity (J) were measured in isolated primary hepatocytes from C57BL/6 normal mice (n = 4−6 per group). (K and L) Metformin-stimulated AMPK activity (K) and lipogenic gene expression (L) were measured in isolated primary hepatocytes from control and L-ROCK1–/– mice fed a normal chow diet (n = 4−8 per group). (M–O) Blood glucose (M), hepatic AMPK activity (N), and lipogenic gene expression (O) were measured in L-CA-ROCK1 mice fed an HFD treated with metformin at 24 weeks of age (n = 6−13 per group). HFD-fed mice (from 6 weeks of age) were treated with metformin for 8 weeks from 16 weeks of age. Values are means ± SEM. *P < 0.05, **P < 0.01 vs. vehicle, †P < 0.05, ‡P < 0.01 vs. control, §P < 0.05 vs. chow, #P < 0.05 vs. HFD by ANOVA with Fisher’s PLSD.

Article Snippet: The membranes were incubated with polyclonal antibodies against ROCK1 (H-85, sc-5560, Santa Cruz Biotechnology), ROCK2 (H-85, sc-5561, Santa Cruz Biotechnology), α2AMPK (Abcam), and actin (C-11, sc-1615, Santa Cruz Biotechnology).

Techniques: Activity Assay, Gene Expression, Isolation, Control

Journal: Journal of Clinical Investigation

Article Title: Rho-kinase/AMPK axis regulates hepatic lipogenesis during overnutrition

doi: 10.1172/jci63562

Figure Lengend Snippet: Figure 8. Regulation of lipogenic gene expression by AMPK. (A) Gene expression of lipogenic enzymes was measured in isolated primary hepatocytes from control and L-ROCK1–/– mice fed a normal chow diet treated with compound C (n = 5–7). (B) Gene expression of lipogenic enzymes was measured in isolated primary hepatocytes from control and L-CA-ROCK1 mice fed a normal chow diet treated with AICAR (n = 3–6). Mice were stud- ied at 8−9 weeks of age. Values are means ± SEM. *P < 0.05, **P < 0.01 vs. vehicle, †P < 0.05, ‡P < 0.01 vs. control by ANOVA with Fisher’s PLSD.

Article Snippet: The membranes were incubated with polyclonal antibodies against ROCK1 (H-85, sc-5560, Santa Cruz Biotechnology), ROCK2 (H-85, sc-5561, Santa Cruz Biotechnology), α2AMPK (Abcam), and actin (C-11, sc-1615, Santa Cruz Biotechnology).

Techniques: Gene Expression, Isolation, Control

Journal: Journal of Clinical Investigation

Article Title: Rho-kinase/AMPK axis regulates hepatic lipogenesis during overnutrition

doi: 10.1172/jci63562

Figure Lengend Snippet: Figure 9. Proposed model for a role of ROCK1 in hepatic metabolism. Upon high-fat feeding, the endocannabinoids 2-AG and AEA are produced and released from hepatic stellate cells of the liver. Once endocannabinoids bind to a cannabinoid receptor (CB1) on the cell surface of hepatocytes, ROCK1 is rapidly activated and subsequently inhibits AMPK, leading to stimulation of the SREBP1c-mediated lipogenic pathway. As a result, hepatic lipid accu- mulates. In contrast, an antidiabetic drug, metformin, suppresses ROCK1 activity but stimulates AMPK activity, which contributes to decreased lipid accumulation in hepatocytes. Thus, we propose a novel signaling pathway for hepatic fatty acid synthesis that is regulated through a ROCK1-dependent mechanism, negatively engaged to AMPK.

Article Snippet: The membranes were incubated with polyclonal antibodies against ROCK1 (H-85, sc-5560, Santa Cruz Biotechnology), ROCK2 (H-85, sc-5561, Santa Cruz Biotechnology), α2AMPK (Abcam), and actin (C-11, sc-1615, Santa Cruz Biotechnology).

Techniques: Produced, Activity Assay

Journal: Molecules

Article Title: KMUP-1 Attenuates Endothelin-1-Induced Cardiomyocyte Hypertrophy through Activation of Heme Oxygenase-1 and Suppression of the Akt/GSK-3β, Calcineurin/NFATc4 and RhoA/ROCK Pathways

doi: 10.3390/molecules200610435

Figure Lengend Snippet: Effects of KMUP-1 on the RhoA/ROCK pathway. H9c2 cells were pretreated with different doses of KMUP-1 for 1 h, followed by co-treatment with 100 nM ET-1 for 30 min. The expression levels of Rho-kinase (ROCK-1 and ROCK-2) and ET-1-induced RhoA translocation were markedly attenuated by KMUP-1 and Fasudil (10 μM). Each value represents the mean ± SEM of three independent experiments, with triplicate determinations in each experiment. # p < 0.01 compared with control; * p < 0.05, ** p < 0.01 compared with ET-1 alone.

Article Snippet: Polyclonal antibody against ROCK-1 was purchased from Chemicon (Temecula, CA, USA).

Techniques: Expressing, Translocation Assay