Fig 2: TNFa increased basal lipolysis and degraded PLIN1, which requires Cathepsin Ba 3T3-L1 adipocytes were individually treated with 10 ng/ml and 50 ng/ml TNFa for 3 h, then media was removed for glycerol measurement. b 3T3-L1 adipocytes were stimulated with 10 ng/ml TNFa for 3 h with or without 1 h CA074 pre-treatment, then media was removed for glycerol measurement. c–d C57BL/6 mice were fed a HFD or standard chow for 24 weeks, then expression levels of PLIN1 in EAT were determined by qRT-PCR c and western blot d (n = 6). e 3T3-L1 adipocytes were treated with the proteasome inhibitor MG-132 (10 µm) or the lysosomal protease inhibitor leupeptin (Leu; 10 µg/ml) for 24 h, then the content of PLIN1 were assessed by western blot. f PLIN1 stability in response to TNFa (10 ng/ml) was evaluated in a cycloheximide (CHX, 100 µg/ml) chasing experiment. Immunoblot of perilipin after 6, 12, 18, and 24 h treatments with CHX in adipocytes. g Adipocytes were stimulated with TNFa (10 ng/ml) in the presence or absence of CA074 treatment (10 µm). Immunoblotting of PLIN1 was perform after treatment with CHX for 6, 12, 18, and 24 h. **p < 0.01, ***p < 0.001. The results are representative of at least three independent repeats in the cell experiments

Fig 3: PLIN1 degradation could be accelerated by the activation of the autophagy–lysosome pathwaya Mature adipocytes infected with lentivirus-mediated control shRNA or shRNA to Becn1 were treated with ABT737 (100 nm) for 12 h. Representative immunoblots of PLIN1 and GAPDH. b Cells were harvested at 0, 6, 12, or 24 h after the addition of CHX (100 µg/ml) with or without ABT737 treatment (100 nm), then the stability of PLIN1 was evaluated using western blot. c–d 8-week-old male C57BL/6 mice were injected intraperitoneally with ABT737 (75 mg/kg) for 14 consecutive days (n = 6), then protein expression of PLIN1 and LC3 in the subcutaneous fat c and epididymal fat d was determined by western blot. *p < 0.05, **p < 0.01, ***p < 0.001. The results are representative of at least three independent repeats in the cell experiments

Fig 4: HSL co-localizes with PLIN1 in human primary adipocytes under lipogenic conditions. Following lipogenic or lipolytic stimulation, cells were double immunolabeled for the detection of PLIN1 (red) and HSL (green). Standard deviation projections from 3D z-stacks of individual adipocytes stimulated with either INS (a,c,e) or ISO (b,d,f), respectively, are shown. (g,h) The effect of PKA on PLIN1 segregation into micro domains was assessed with hormone stimulations in presence of the PKA inhibitor H-89. PLIN1 staining is shown in red and DAPI nuclei stain in blue. Shown are: (g) INS-stimulated in presence 25 µM H-89; (h) ISO-stimulated in presence of H-89. Scale bars are 25 µm.

Fig 5: Nestin-Cre and Nestin-GFP identify perivascular cells in WAT. (A) Schematic of the genetic tools in Nes-GFP; Nes-Cre; R26-Tomato dual-reporter mice used in this figure. GFP and Cre are expressed from distinct nestin-driven transgenes. Cre acts on a Cre/lox-dependent R26 knock-in fluorescent Tomato reporter, which serves as a lineage trace. (B) Epifluorescence of Nes-Cre/Tomato lineage tracing in visceral WAT, imaged by whole-mount with isolectin-IB4 staining for capillary endothelial cells. (C) Measurement of the distance between DAPI+ nuclei of individual Nes-GFP+ cells (n = 167) and the nearest IB4+ capillary membrane, as shown in the example at the right. A distance <10 µm means the cell is on the abluminal surface of the capillary. (D) Epifluorescence of Nes-GFP and Nes-Cre/Tomato plus immunofluorescence staining of Myh11 in vascular smooth muscle cells. The GFP/Tomato reporters are coexpressed in pericyte-like cells (arrowhead). Tomato also identifies adventitial cells in which Nes-GFP is not expressed (arrow). (E–I) Epifluorescence of Nes-GFP and Nes-Cre/Tomato plus IB4 labeling for endothelial cells (E), immunofluorescence staining of Cspg4 or PDGFRß for pericytes (F,G), immunofluorescence staining of collagen IV for basement membrane (H), and immunofluorescence staining of PDGFRa (I). (J) Epifluorescence of Nes-GFP and Nes-Cre/Tomato plus immunofluorescence staining of Perilipin (PLIN1) for adipocytes. Tomato identifies rare adipocytes (arrow) that do not express Nes-GFP. A Tomato+GFP+ pericyte-like cell is also shown (arrowhead). Bars: B, 100 µm; D, 30 µm; E–I, 20 µm; J, 30 µm.