Fig 2: The effect of KPL combined with ABT-199 on apoptosis of HepG2 cells. (A) Representative images of cell apoptosis in various groups. Different concentrations of KPL with or without 2 μM ABT-199 were treated on HepG2 cells for 24 h, and then the apoptosis was detected by Hoechst 33342/FITC-Annexin V/PI triple staining. The images were collected by an Arrayscan VT1 HCS reader. Merged images were the composition of images of the same field from three different channels. (B) The effects of various concentrations of KPL with or without ABT-199 on the nuclear area. (C) Fluorescence intensity of FITC Annexin V staining in HepG2 cells treated by various concentrations of KPL with or without ABT-199. (D) Fluorescence intensity of PI staining in HepG2 cells treated by various concentrations of KPL with or without ABT-199. Values are means ± SD (n = 12). **p < 0.01, ***p < 0.001 versus negative control group; ΔΔ p < 0.01, ΔΔΔ p<0.001 versus ABT-199 alone group; # p < 0.05, ### p < 0.001 versus KPL 100 μM group; ○ p < 0.05, ○○○ p < 0.001 versus KPL 200 μM group.

Fig 3: Overexpression of MB in imBA cells increases mitochondrial respiration and acute response to adrenergic stimulation. (A) Expression of hMB in stably transfected imBA_Ctrl and imBA_hMB adipocytes at day 0 and day 8 of differentiation. Gene expression is relative to imBA_Ctrl at day 0 and normalized to Nono. (B) Western blot analysis of total (mouse and human) MB expression and (C) ELISA‐based quantification of hMB protein in differentiated imBA_Ctrl and imBA_hMB adipocytes. (D) Quantification of lipid accumulation in imBA_Ctrl and imBA_hMB adipocytes during differentiation. (E) Fluorescence microscopy (left panel) and droplet size measurements (right panel) of AdipoRed‐stained lipid droplets in differentiated imBA_Ctrl and imBA_hMB adipocytes (day 8). (F) Time‐resolved OCR of differentiated imBA_Ctrl and imBA_hMB adipocytes measured by Seahorse (representative experiment, n = 6/6). (G) Quantification of basal respiration, proton leak, ATP production, acute response to CL316,243 (CL), maximum and spare respiratory capacity and non‐mitochondrial respiration of samples in panel F. (H) Lipolytic (Adrb3, Atgl, Hsl, Plin1), lipogenic (Chrebpb, Acly, Acacb, Elovl6, Fasn, Scd1), fatty acid (FA) transporter (Fatp1, Fatp4), and mitochondrial (Ndufa2, Sdhb, Cycs, CoxVI, Atp5g1) gene expression in differentiated imBA_Ctrl and imBA_hMB adipocytes. (I) Basal and CL‐induced thermogenic (Ucp1, Pgc1a, Cd36) gene expression in differentiated imBA_Ctrl and imBA_hMB adipocytes. (J) Western blot analysis and (K) quantification of basal and CL‐induced PKA‐activation in differentiated imBA_Ctrl and imBA_hMB adipocytes. Upper panel: anti‐phospho‐PKA substrate antibody; lower panel: anti‐ACTB antibody. (L) Measurements of basal and CL‐induced glycerol, (M) free fatty acid (free FA) and (N) lactate release from differentiated imBA_Ctrl and imBA_hMB adipocytes. Data are presented as mean ± SEM of at least two (L,M) or three independent experiments. Statistical significance was evaluated by two‐way ANOVA with Šídák's (A,C) or Tukey's (I–N) post hoc test or unpaired t‐test (E), or multiple unpaired t‐tests corrected by the Holm–Šídák method (G) or uncorrected (H). *p‐value < .05, **p‐value < .01, ***p‐value < .001. Scale bar: 50 μm.