Journal: iScience

Article Title: Induction of adipocyte thermogenic program by Rho-associated coiled-coil containing kinase 2

doi: 10.1016/j.isci.2026.115225

Figure Lengend Snippet: ROCK2 regulates mitochondrial gene expression and function in subcutaneous WAT Male control and AdR2KO mice were housed at RT or 4 °C for 3 days, at which point sWAT was harvested and subjected to mitochondrial PCR array. (A) Heatmap shows changes in mitochondrial gene expression in sWAT ( n = 4 mice per group). (B–H) The oxygen consumption rate (OCR) in the sWAT of male control and AdR2KO mice housed at RT or 4 °C for 3 days was measured with a Seahorse XFe24 Analyzer ( n = 4 mice per group). (B) Oligomycin (an ATPase inhibitor), FCCP (a chemical uncoupler) and Rot/AA (rotenone/antimycin A, mitochondrial complex I and III inhibitors) were added sequentially to determine the rates of (C) basal, (D) ATP-linked, (E) maximal, (F) spare, (H) non-mitochondrial respiration, and (G) proton leak. p values were determined by one-way ANOVA with Tukey’s multiple comparisons test. All data are presented as the mean ± SEM of at least three independent biological replicates. See also and .

Article Snippet: ROCK2 siRNA (h) , Santa Cruz Biotechnology , Cat#sc-29474.

Techniques: Gene Expression, Control

Journal: iScience

Article Title: Induction of adipocyte thermogenic program by Rho-associated coiled-coil containing kinase 2

doi: 10.1016/j.isci.2026.115225

Figure Lengend Snippet: ROCK activity increases after cold exposure in a ROCK2-dependent manner (A) The mRNA levels of Rock isoforms in interscapular brown adipose tissue (iBAT), inguinal subcutaneous white adipose tissue (sWAT), and epididymal (epiWAT) from 8-week-old male wild-type (WT) C57BL/6J mice at room temperature (RT) ( n = 6 mice) or after exposure to 4 °C for 3 h ( n = 5 mice). (B) The ROCK activity was indicated by the ratio of phosphorylated to total myosin-binding subunit (MBS) of myosin phosphatase, a direct downstream target of ROCK. Western blotting and quantification of ROCK1, ROCK2, phosphorylated MBS (pMBS), and total MBS (tMBS) in iBAT, sWAT, and epiWAT ( n = 4 mice per time point) from male WT mice housed at 4 °C for 0, 1, 3, or 6 h (C and D) Western blotting and quantification of ROCK1, ROCK2, phosphorylated and total MBS in sWAT from male WT and Rock2 +/− mice housed at RT or 4 °C for 6 h ( n = 4 mice per genotype). (E) Western blotting and quantification of ROCK1, ROCK2, pMBS, and tMBS in iBAT, sWAT, and epiWAT from male Adipoq-Cre +/− Rock2 +/+ (control) and Adipoq-Cre +/− Rock2 flox/flox (AdR2KO) mice housed at RT or 4 °C for 3 h ( n = 4 mice per group). (F) Western blotting and quantification of ROCK1, ROCK2, pMBS, and tMBS in sWAT from male control and AdR2KO mice housed at RT or 4 °C for 3 days ( n = 4 mice per group). The immunoblot images are representative of at least three independent experiments. p values were determined by two-tailed Student’s t test (A, C, and F) or one-way ANOVA with Tukey’s multiple comparisons test (B, D, E, and F). All data are presented as the mean ± SEM of at least three independent biological replicates.

Article Snippet: ROCK2 siRNA (h) , Santa Cruz Biotechnology , Cat#sc-29474.

Techniques: Activity Assay, Binding Assay, Western Blot, Control, Two Tailed Test

Journal: iScience

Article Title: Induction of adipocyte thermogenic program by Rho-associated coiled-coil containing kinase 2

doi: 10.1016/j.isci.2026.115225

Figure Lengend Snippet: ROCK2 mediates the beiging of subcutaneous WAT The mRNA levels of (A) brown fat-selective genes ( Ppargc1a , Ucp1 , Prdm16 , and Cidea ) and (B) general adipocyte genes ( Adipoq and Slc2a4 ) in sWAT from male control and AdR2KO mice housed at RT or 4 °C for 3 days ( n = 8 mice per group). (C) Immunohistochemistry for UCP1 in sections of sWAT from male control and AdR2KO mice housed at RT or 4 °C for 3 days ( n = 3 mice per group). High-magnification images are shown in the boxed square. The scale bars are 5 mm and 100 μm in the low- and high-magnification images, respectively. (D) Western blotting and quantification of UCP1 in the sWAT of male control and AdR2KO mice housed at RT or 4 °C for 3 days ( n = 4 mice per group). (E) The mRNA levels of brown fat-selective genes ( Ppargc1a and Ucp1 ) in epiWAT from male control and AdR2KO mice housed at RT or 4 °C for 3 days ( n = 6 mice per group). (F) Immunohistochemistry for UCP1 in sections of epiWAT from male control and AdR2KO mice housed at RT or 4 °C for 3 days ( n = 3 mice per group). Scale bars, 100 μm. The histological and immunoblot images are representative of at least three independent experiments. p values were determined by one-way ANOVA with Tukey’s multiple comparisons test. All data are presented as the mean ± SEM of at least three independent biological replicates.

Article Snippet: ROCK2 siRNA (h) , Santa Cruz Biotechnology , Cat#sc-29474.

Techniques: Control, Immunohistochemistry, Western Blot

Journal: iScience

Article Title: Induction of adipocyte thermogenic program by Rho-associated coiled-coil containing kinase 2

doi: 10.1016/j.isci.2026.115225

Figure Lengend Snippet: ROCK2-mediated thermogenic gene expression may occur through the JNK/FOXO1/UCP1 axis (A) Western blotting and quantification of FOXO1 in sWAT from male WT mice housed at room or cold temperature for 3 h ( n = 4 mice per group). (B) Ucp1 expression in sWAT from male WT mice housed at 4 °C for 0, 3, 6, or 12 h ( n = 4 mice). (C) Ucp1 expression in sWAT harvested from control mice and then treated with 40 μM isoproterenol (Iso) and/or 4 μM FOXO1 inhibitor (AS1842856) for 6 h ( n = 4 wells per group). (D) Foxo1 expression in sWAT from male control and AdR2KO mice housed at RT or cold for 3 h ( n = 4 mice). (E) Western blotting and quantification of nuclear FOXO1 (nFOXO1), cytoplasmic FOXO1 (cFOXO1), and total FOXO1 (tFOXO1) in sWAT from male control and AdR2KO mice housed at RT or cold for 3 h ( n = 3 mice). (F) Western blotting and quantification of phosphorylated JNK (pJNK) and total JNK (tJNK) in sWAT from male WT mice housed at RT or cold for 3 h ( n = 3 mice). (G) Foxo1 expression in sWAT harvested from control mice and then treated with 40 μM isoproterenol (Iso) and/or 100 μM JNK inhibitor (SP600125) for 6 h ( n = 6 wells per group). (H) Western blotting and quantification of pJNK and tJNK in sWAT from control and AdR2KO mice housed at RT or cold for 3 h ( n = 3 mice). (I) Foxo1 and (J) Ucp1 expression in differentiated primary adipocytes from sWAT of control and AdR2KO mice. After differentiation for 8 days, mature adipocytes were treated with or without 10 μM isoproterenol (Iso), 1 μM AS1842856, or 50 μM SP600125 for 6 h n = 5–6 wells in (I); n = 3–8 wells in (J). (K) The mRNA levels of Mapk8 (encodes JNK1), Foxo1 , Ppargc1a , and Ucp1 in mature mouse adipocyte cells, 3T3-L1. Adipocytes were transfected with siRNAs for 72 h and then treated with 10 μM isoproterenol (Iso) for 6 h ( n = 6 wells per group). (L) The mRNA levels of Foxo1 , Ppargc1a , and Ucp1 in differentiated mouse primary adipocytes from sWAT of control and AdR2KO mice. Adipocytes were transduced with low ( Foxo1L ) and high ( Foxo1H ) MOI of AAV-8 containing mouse Foxo1 plasmid for 7 days and then treated with 20 μM isoproterenol (Iso) for 16 h ( n = 8 wells per group). The immunoblot images are representative of at least three independent experiments. p values were determined by the two-tailed Student’s t test (A and F) or one-way ANOVA with Tukey’s multiple comparisons test (B-E and G-L). All data are presented as the mean ± SEM of at least three independent biological replicates. See also and . AS: AS1842856; SP: SP600125.

Article Snippet: ROCK2 siRNA (h) , Santa Cruz Biotechnology , Cat#sc-29474.

Techniques: Gene Expression, Western Blot, Expressing, Control, Transfection, Transduction, Plasmid Preparation, Two Tailed Test

Journal: iScience

Article Title: Induction of adipocyte thermogenic program by Rho-associated coiled-coil containing kinase 2

doi: 10.1016/j.isci.2026.115225

Figure Lengend Snippet: ROCK2 contributes to adaptive thermogenesis in interscapular BAT (A) Representative thermal images of male control ( n = 7 mice) and AdR2KO ( n = 6 mice) mice and quantification of the surface temperatures of the back and interscapular regions. The thermal images are representative of at least six independent biological replicates. (B) Immunohistochemistry for UCP1 in the iBAT of male control and AdR2KO mice housed at RT or 4 °C for 3 days ( n = 3 mice per group). High-magnification images are shown in the boxed square. The scale bars are 5 mm and 100 μm in the low- and high-magnification images, respectively. (C) Western blotting and quantification of UCP1 in the iBAT of male control and AdR2KO mice housed for 3 days at RT or 4 °C ( n = 4 mice per group). (D) Brown fat-selective gene ( Ppargc1a , Ucp1 , and Cidea ) and general adipocyte gene ( Adipoq and Slc2a4 ) expression in the iBAT of male control and AdR2KO mice housed at RT or 4 °C for 3 days ( n = 6 mice per group). The immunohistochemistry and immunoblot images are representative of at least three independent experiments. p values were determined by one-way ANOVA with Tukey’s multiple comparisons test (A, C, and D). All data are presented as the mean ± SEM of at least three independent biological replicates.

Article Snippet: ROCK2 siRNA (h) , Santa Cruz Biotechnology , Cat#sc-29474.

Techniques: Control, Immunohistochemistry, Western Blot, Expressing

Journal: iScience

Article Title: Induction of adipocyte thermogenic program by Rho-associated coiled-coil containing kinase 2

doi: 10.1016/j.isci.2026.115225

Figure Lengend Snippet: ROCK2 deletion decreased the oxygen consumption rate in AdR2KO mice after cold exposure The whole-body oxygen consumption rate (VO 2 ), carbon dioxide production rate (VCO 2 ), and respiratory exchange rate (RER) in 10- to 12-week-old male control and AdR2KO mice housed at (A) room temperature (RT) or (B) cold (4 °C) for 72 h ( n = 6 mice per group). (C) The mRNA expression of brown fat-selective genes ( Ppargc1a , Ucp1 , and Cidea ) and general adipocyte genes ( Adipoq and Slc2a4 ) in the sWAT of male control and adipocyte-specific constitutively active ROCK (AdcaRK) mice ( n = 6 mice per group). (D) Immunohistochemistry for UCP1 in sections of sWAT from control and AdcaRK mice housed at RT or 4 °C for 3 days ( n = 3 mice per group). High-magnification images are shown in the boxed squares. The scale bars are 5 mm and 100 μm in the low- and high-magnification images, respectively. The images are representative of three independent experiments. (E) The mRNA level of Ucp1 in sWAT harvested from control and AdcaRK mice and then treated with 40 μM isoproterenol (Iso) and 4 μM FOXO1 inhibitor (AS1842856) for 6 h ( n = 6 wells per group). (F) The mRNA levels of Foxo1 and Irf4 in sWAT from male control and AdcaRK mice housed at RT or cold for 3 days ( n = 6 mice per group). p values were determined by two-tailed Mann-Whitney U tests (A and B) or one-way ANOVA with Tukey’s multiple comparisons test (C, E, and F). All data are presented as the mean ± SEM of at least three independent biological replicates. See also . AS: AS1842856.

Article Snippet: ROCK2 siRNA (h) , Santa Cruz Biotechnology , Cat#sc-29474.

Techniques: Control, Expressing, Immunohistochemistry, Two Tailed Test, MANN-WHITNEY

Journal: iScience

Article Title: Induction of adipocyte thermogenic program by Rho-associated coiled-coil containing kinase 2

doi: 10.1016/j.isci.2026.115225

Figure Lengend Snippet: The metabolic phenotypes in modulating ROCK2 in adipocytes (A) Body weight, (B) adipose depot mass to body weight ratio, (C) oral glucose tolerance test, ∗ p < 0.05, and (D) total area under curve (AUC) of (C) in 20- to 24-week-old male control ( n = 5 mice) and AdR2KO ( n = 7 mice) mice housed at room temperature. (E) Body weight change, (F) adipose depot mass to body weight ratio, (G) oral glucose tolerance test, ∗ p < 0.05, and (H) total area under curve of (G) in 8- to 10-week-old male control ( n = 5 mice) and AdcaRK ( n = 6 mice) mice housed at 4 °C for 3 days. (I) Body weight of control, AdR2KO, and AdcaRK mice fed on a high-fat diet (HFD) for 19 weeks starting at 14 weeks of age ( n = 5 mice per group). ∗ p < 0.05 compared to week 1 in the same genotype. † p < 0.05 compared to control mice. p values were determined by two-tailed unpaired t tests (A to I). All data are presented as the mean ± SEM of at least three independent biological replicates.

Article Snippet: ROCK2 siRNA (h) , Santa Cruz Biotechnology , Cat#sc-29474.

Techniques: Control, Two Tailed Test