Figure 7. Deletion of FNIP1 activates Ca... | Rockefeller University Press

Figure 7.

Deletion of FNIP1 activates Ca2+-dependent thermogenic program in white adipocytes. (A–C) Ca2+ transients in differentiated white adipocyte isolated from iWAT of male WT and FNIP1 KO mice. Ca2+ dynamics were monitored using Fluo-4 AM in cells following exposure to 1 μM NE. n = 58–81 cells per group from five independent experiments. (A) Representative time-lapse images of indicated adipocytes at different time intervals after NE stimulation. Scale bars: 20 μm. (B) Quantitative analysis of the Fluo-4 AM fluorescence intensity (Ft/F0). (C) Quantitation of amplitude, FDHM, and time constant Tau of Ca2+ transients. (D–F) Ca2+ transients in differentiated white adipocyte isolated from iWAT of male GCaMP6f transgenic and GCaMP6f; FNIP1 KO mice. n = 64–70 cells per group from five independent experiments. (D) Representative time-lapse images of indicated adipocytes at different time intervals after NE stimulation. Scale bars: 50 μm. (E) Quantitative analysis of the GCaMP6f fluorescence intensity (Ft/F0). (F) Quantitation of amplitude, FDHM, and time constant Tau of Ca2+ transients. (G and H) OCRs in differentiated white adipocyte isolated from iWAT of male WT and FNIP1 KO mice. Basal OCR was first measured, followed by administration of 2 μM oligomycin (to inhibit ATP synthase), uncoupler FCCP (2 μM), or rotenone/antimycin (Rot/A; 1 μM) as indicted. n = 3 independent experiments done with 5–7 biological replicates. (H) Cells were pre-incubated with 10 μM BAPTA-AM for 2 h as indicated in the key. (I and J) Gene expression (qRT-PCR) in differentiated white adipocyte isolated from iWAT of male WT and FNIP1 KO mice. Cells were treated for 4 h with DMSO (vehicle), 10 μM FSK, 20 μM CL, 10 μM BAPTA-AM, FSK + BAPTA-AM, or CL + BAPTA-AM as indicated in the key. n = 3 independent experiments. Values represent mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001. P value was determined using two-tailed unpaired Student’s t-test (B, C, E, and F), one-way ANOVA and Fisher’s LSD post-hoc test (I and J), or two-way ANOVA and Bonferroni post-hoc test (G and H). Data are representative of at least three independent experiments (A–J).

Deletion of FNIP1 activates Ca ²⁺ -dependent thermogenic program in white adipocytes. (A–C) Ca²⁺ transients in differentiated white adipocyte isolated from iWAT of male WT and FNIP1 KO mice. Ca²⁺ dynamics were monitored using Fluo-4 AM in cells following exposure to 1 μM NE. n = 58–81 cells per group from five independent experiments. (A) Representative time-lapse images of indicated adipocytes at different time intervals after NE stimulation. Scale bars: 20 μm. (B) Quantitative analysis of the Fluo-4 AM fluorescence intensity (F_t/F₀). (C) Quantitation of amplitude, FDHM, and time constant Tau of Ca²⁺ transients. (D–F) Ca²⁺ transients in differentiated white adipocyte isolated from iWAT of male GCaMP6f transgenic and GCaMP6f; FNIP1 KO mice. n = 64–70 cells per group from five independent experiments. (D) Representative time-lapse images of indicated adipocytes at different time intervals after NE stimulation. Scale bars: 50 μm. (E) Quantitative analysis of the GCaMP6f fluorescence intensity (F_t/F₀). (F) Quantitation of amplitude, FDHM, and time constant Tau of Ca²⁺ transients. (G and H) OCRs in differentiated white adipocyte isolated from iWAT of male WT and FNIP1 KO mice. Basal OCR was first measured, followed by administration of 2 μM oligomycin (to inhibit ATP synthase), uncoupler FCCP (2 μM), or rotenone/antimycin (Rot/A; 1 μM) as indicted. n = 3 independent experiments done with 5–7 biological replicates. (H) Cells were pre-incubated with 10 μM BAPTA-AM for 2 h as indicated in the key. (I and J) Gene expression (qRT-PCR) in differentiated white adipocyte isolated from iWAT of male WT and FNIP1 KO mice. Cells were treated for 4 h with DMSO (vehicle), 10 μM FSK, 20 μM CL, 10 μM BAPTA-AM, FSK + BAPTA-AM, or CL + BAPTA-AM as indicated in the key. n = 3 independent experiments. Values represent mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001. P value was determined using two-tailed unpaired Student’s t-test (B, C, E, and F), one-way ANOVA and Fisher’s LSD post-hoc test (I and J), or two-way ANOVA and Bonferroni post-hoc test (G and H). Data are representative of at least three independent experiments (A–J).