phospho foxo1  (Cell Signaling Technology Inc)

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: The PVDF membranes were blocked with 5% nonfat dry milk (Research Products International; M17200 ) in TBST for 1 hour and then incubated overnight at 4°C with primary antibodies against ROCK1 (BD Biosciences; 611136), ROCK2 (BD Biosciences; 610623), phospho-Thr 850 MBS (Millipore; 36-003), total MBS (BioLegend; 925101), UCP1 (Abcam; ab23841), phospho-FOXO1 (Ser 256 , Cell Signaling; 9461), FOXO1 (Cell Signaling; 2880), phospho-SAPK/JNK (Thr 183 /Tyr 185 , Cell Signaling; 9251), phospho-AKT (Ser473, Cell Signaling Technology; 4060), AKT (Cell Signaling; 9272), total JNK (SAPK/JNK, Cell Signaling; 9252), Lamin A/C (Cell Signaling; 4777), and GAPDH (Cell Signaling; 2118).

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 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: The PVDF membranes were blocked with 5% nonfat dry milk (Research Products International; M17200 ) in TBST for 1 hour and then incubated overnight at 4°C with primary antibodies against ROCK1 (BD Biosciences; 611136), ROCK2 (BD Biosciences; 610623), phospho-Thr 850 MBS (Millipore; 36-003), total MBS (BioLegend; 925101), UCP1 (Abcam; ab23841), phospho-FOXO1 (Ser 256 , Cell Signaling; 9461), FOXO1 (Cell Signaling; 2880), phospho-SAPK/JNK (Thr 183 /Tyr 185 , Cell Signaling; 9251), phospho-AKT (Ser473, Cell Signaling Technology; 4060), AKT (Cell Signaling; 9272), total JNK (SAPK/JNK, Cell Signaling; 9252), Lamin A/C (Cell Signaling; 4777), and GAPDH (Cell Signaling; 2118).

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: 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: anti-Phospho-Ser 256 FOXO1 , Cell Signaling Technology , Cat# 9461; RRID: AB_329831.

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 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: anti-Phospho-Ser 256 FOXO1 , Cell Signaling Technology , Cat# 9461; RRID: AB_329831.

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