Fig 2: Increased lipogenesis and decreased lipolysis were observed in subcutaneous adipose tissue of Asc-/- mice fed with HFD. (a) Body weight change of LM (n = 8/group) and Asc knockout (Asc-/-) mice (n = 10/group), fed with HFD (12 weeks). (b) SAT groups are as described in (a). (c) Fat index (ratio of SAT weight to whole body weight of indicated mice. (d) H&E and quantification of SAT cell size, scale bar 50 µm. (e,f) GTT and ITT analysis of indicated mice. (g) In indicated mice, plasma concentrations of triglyceride (TG), total cholesterol (TC), and free fatty acids (NEFA) at baseline. (h) mRNA expression of lipogenesis genes in SAT from indicated mice. (i) Western blot analysis of lipogenesis (ACC, FASN, FABP4) and lipolysis (p-HSL, HSL, ATGL) proteins in SAT. All the mice were male and fed with HFD for 12 weeks if not indicated otherwise. (n = 8–10/group) (Red-LM mice, Blue-Asc-/- mice). * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

Fig 3: 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 4: ASC defection promoted lipid accumulation through p53/AMPKa axis. (a) Protein expression of phosphor-p53, p53, phosphor-AMPKa, and AMPKa in SAT of LM and Asc-/- mice fed with HFD (12 weeks). (b) Protein expression of phosphor-p53, p53, phosphor-AMPKa, and AMPKa in primary SVF cells separated from SAT of LM and Asc-/- mice (n = 3 per group). (c) Protein expression of phosphor-p53, p53, phosphor-AMPKa, and AMPKa in primary SVF cells separated from WT mice SAT transfected with ASC siRNA (n = 3 per group). (d) Protein expression of phosphor-p53, p53, phosphor-AMPKa, and AMPKa in primary SVF cells separated from LM and Asc-/- mice SAT, with AICAR or Kevetrin treatment (n = 3 per group). (e) Protein expression of lipogenesis (ACC, FASN, FABP4) and lipolysis (p-HSL, HSL, ATGL) in primary SVF cells separated from LM and Asc-/- mice SAT, with AICAR or Kevetrin treated (n = 3 per group). (f) Oil Red O staining of the 6th day under lipogenesis inducement treated in primary SVF cells, separated from LM and Asc-/-- mice, with AICAR or Kevetrin, treated (n = 3 per group), scale bar 200 µm. (g) Protein expression of phosphor-p53, p53, phosphor-AMPKa, and AMPKa in primary SVF cells separated from WT mice, with ASC siRNA, AICAR, or Kevetrin treatment (n = 3 per group). (h) Protein expression of lipogenesis (ACC, FASN, FABP4) and lipolysis (p-HSL, HSL, ATGL) in primary SVF cells separated from WT mice, with ASC siRNA, AICAR, or Kevetrin treated (n = 3 per group). (i) Oil Red O staining on the 6th day under lipogenesis inducement in primary SVF cells separated from WT mice, with ASC siRNA, AICAR, or Kevetrin treatment (n = 3 per group). (AICAR was used at 500 µM for 5 h, Kevetrin was used at 340 µM for 24 h), and the scale bar was 200 µm. Data are presented as means ± SD from three independent experiments.