Fig 2: SRA inhibits PPAR?-mediated ATGL promotor driven luciferase activity.Cells were transfected with -3000/+ 1-LUC ATGL construct (200 ng) and pRL-TK-Renilla (10 ng). (a–d) PPAR? expression vector was cotransfected. 36 h after transfection, cells were treated with rosiglitazone (1 µM) for 24 h before harvesting. (a) HepG2 cells were transfected by PPAR? expression vector with the dose indicated. (b) pSCT-SRA or control expression vector (200 ng) plus PPAR? expression vector (3 ng) were transfected into HepG2 cells. (c) Similar to (b), except with AS1842856 (1 µM) treatment immediately after transfection. (d) Hepa1-6 cells were infected with shRNA against SRA (shSRA) or scrambled shRNA as control (shControl). 12 h later, PPAR? expression vector (3 ng) was transfected into Hepa1-6 cells. AS1842856 (1 µM) treatment was started immediately after transfection. Subsequent luciferase assays were performed 72 h after infection. (e) HepG2 cells were transfected by pSCT-SRA or control expression vector (200 ng), followed by T0070907 (0.4 µM) treatment immediately after transfection. 60 h later, cells were harvested for luciferase assay. (f) Hepa1-6 cells were infected with shRNA against SRA (shSRA) or scrambled shRNA as control (shControl). 12 h later, cells were treated with T0070907 (0.4 µM) for 60 h before harvesting. ATGL promoter driven luciferase activity were normalized to Renilla luciferase activity and presented as mean ± SE. Data are expressed as fold-change relative to the level of control. *p < 0.05.

Fig 3: Generation and characterization of AKO, cHSL, and AKO+cHSL mice. A: Protein expression of ATGL and HSL in the cardiac ventricle of Wt, AKO, cHSL, and AKO+cHSL (A+H) mice, as determined via Western blotting using specific antibodies against ATGL or HSL: 10 µg of the protein sample was subjected to SDS-PAGE, transferred to nitrocellulose membranes, and probed with an appropriate antibody. The graphs present the densitometric analysis of protein expression normalized to that of GAPDH. Values are presented as the mean ± SEM of four mice/group. B: Phosphorylation of HSL using specific antibodies against Ser563, Ser565, and Ser660. Twenty micrograms (Wt and AKO) or 3.3 µg (cHSL and AKO+cHSL) of the protein sample was subjected to SDS-PAGE, transferred to nitrocellulose membranes, and probed with an appropriate antibody. The graphs present the densitometric analysis of phosphorylated HSLs normalized to total HSL expression. A + H denotes AKO+cHSL mice. Values are presented as the mean ± SEM of four mice/group. C: Cardiac TAG hydrolase activity in Wt, AKO, cHSL, AKO+cHSL (A+H), and heterozygous AKO (hetero AKO) mice. Cardiac ventricles were homogenized in TES buffer containing a protease inhibitor cocktail, and the supernatants were subjected to the assay. Values are presented as the mean ± SEM of four mice/group. D: Cardiac TAG content in Wt, AKO, cHSL, and AKO+cHSL (A+H) mice. Lipids were extracted from cardiac ventricles and quantified as per methods described in the Materials and Methods section. Values are presented as mean ± SEM of five (Wt, cHSL, and AKO+cHSL) or four (AKO) mice in each group. E: Representative light (upper panels) and electron (lower panels) micrographs of ventricular tissues derived from Wt, AKO, cHSL, and AKO+cHSL mice. The scale bars represent 100 µm (upper panels) or 5.0 µm (lower panels). Five mice per group, three to five sections per mouse were analyzed, and representative images have been presented. *P < 0.05 and **P < 0.01 versus Wt mice; †P < 0.05 and ††P < 0.01 versus AKO mice.

Fig 4: Ablation of ASC in stromal vascular fractions (SVF) cells improved the lipogenesis but impaired lipolysis. (a–d) Primary SAT SVF cells of LM and Asc-/- mice (Red-LM mice, Blue-Asc-/- mice). (a) Oil Red O staining of the 6th day under lipogenesis inducement treated primary SAT SVF cells, separated from LM and Asc-/- mice, scale bar 200 µm. (b) The expression of lipogenesis genes was analyzed by RT-qPCR. (c) Expression of lipogenesis (ACC, FASN, FABP4) and lipolysis (p-HSL, HSL, ATGL) protein in indicated cells. (d–h) Primary SAT SVF cells separated from WT mice were transfected with siRNA targeting for ASC on the 4th day (Blue-NC, Yellow-si Asc). (d,e) mRNA and protein level of ASC in indicated cells. (f) Oil Red O staining of the 6th day under lipogenesis inducement treated in indicated cells, scale bar 200 µm. (g) Expression of lipogenesis genes in indicated cells. (h) Expression of lipogenesis (ACC, FASN, FABP4) and lipolysis (p-HSL, HSL, ATGL) protein in indicated cells. Data are presented as means ± SD from three independent experiments. * p < 0.05 and ** p < 0.01.

Fig 5: A. ATGL (n = 8) and CGI-58 (n = 7) protein content in the three skeletal muscles (SOL, RG, and WG and heart).Ponceau stain for total protein was used as a loading control. Data are reported as mean±SE and bars with the same letter are not significantly different within the skeletal muscles (p<0.05). Insets: representative blots: lane 1, WG (white gastrocnemius); lane 2, RG (red gastrocnemius); lane 3, SOL (soleus). B. ATGL and CGI-58 western blot protein bands compared to representative Ponceau bands as equal loading control.