Perilipin 1

Adipose tissues were fixed and processed for histological analysis, as previously described [7 (link)]. Paraffin sections (5-μm thick) were subjected to immunohistochemical analysis, as previously described [7 (link)]. The antibodies used for immunochemical detection were anti-UCP1 antibody (rabbit, 0.5 μg/ml, Alpha Diagnostic International), perilipin 1 (rabbit, 1:100, Cell Signaling), and tyrosine hydroxylase antibody (mouse, 1:400, Merck Millipore). Secondary antibodies used were goat anti-rabbit-Alexa Fluor 488 and goat anti-mouse-Alexa Fluor 594 (1:500, Invitrogen, Molecular Probes). IgG controls (normal rabbit IgG, Santa Cruz) were used as negative controls for IHC analysis, when the information on the concentration of primary antibodies was available (Additional file 1: Figure S1). Otherwise, the omission of primary antibody was used as a negative control. DAPI (Sigma) was used as a nuclear counter stain.

Antibodies against Peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT/enhancer binding protein-α (C/EBPα), fatty acid binding protein 4 (FABP4), and lipoprotein lipase (LPL) were from Abcam (Cambridge, MA, USA). Antibodies against SPRY4 and GAPDH were from Proteintech (Wuhan, China). Antibodies against phosphorylated (p)-ERK1/2, ERK1/2 and Perilipin 1 were from Cell Signalling Technology (Danvers, MA, USA). U0126 was from Selleck Chemicals (Houston, TX, USA).

For Western blot analysis, cell lysates mixed with 6× loading buffer (Beyotime, Shanghai, China) were denatured at 100 °C for 5 min and separated on a 10% SDS-PAGE gel(Bio-Rad, Hercules, CA, USA). The proteins were transferred to a nitrocellulose (NC) membrane (Millipore, Boston, MA, USA). Then, the membrane was blocked for 2 h and incubated overnight with the appropriate primary antibody (Perilipin-1, Cell Signaling Technology, Danvers, MA, USA) at 4 °C. The next day, the membrane was incubated with an HRP-conjugated secondary antibody (ZSGB-Bio, Beijing, China) for 1 h at room temperature. Specific protein bands were visualized using the ECL Plus Detection Kit (Beyotime, Shanghai, China) with a chemiluminescence system (Sagecreation, Beijing, China) and ImageQuant LAS 500 system (GE, Piscataway, NJ, USA).

The following Cell Signaling Technology antibodies were used: ATM (13934), perilipin‐1 (#9349), ACC (#3662), ACLY (#13390), pACLY (#4331), carbohydrate response element binding ptotein (ChREBP) (#58069), carnitine palmitoyl transferase 1 (CPT1) (#12252), stearyl‐coenzyme A desaturase (SCD1) (#2794), Akt (#4691 or #9272), pAktS473 (#9271), pAktT308 (#13038), phospho‐mammalian target of rapamycin (pmTOR) (#5536), pS6 (#2215), pp70S6K (#9234), pH2Ax (#9718), pH3 (53348), cyclin B1 (#12231), phospho‐insulin receptor (pINSR) (#3026), cleaved caspase‐3 (#9661), actin (#5125), PTEN (#9188). Plin1 (ab61682), Plin2 (ab108323 and ab52356), Plin3 (ab47638), Plin4 (ab234752), Plin5 (ab222811), sterol regulatory‐element binding protein (SREBP) (ab28481), Mad2 (ab97777), GFP (ab290), pericentrin (ab4448), CD36 (ab133625), tubulin (ab7291), and valosin‐containing protein (VCP) (ab11433) were ordered from abcam, P350 (ATA‐AMAB91164‐100) from Biozol, Trip13 (19602‐1‐AP) from Proteintech, and INSR (sc‐711), FASN (sc‐20140) from SantaCruz Biotech.

Freshly collected adipose tissues were fixed in 4% paraformaldehyde overnight at 4°C and rinsed with PBS. After serial dehydration in ethanol, tissues were embedded in paraffin and sectioned into 5-μm-thick sections for staining with the HE solution (Servicebio, Cat# G1003). For Tunel staining, sections were deparaffinized and rehydrated by serial xylene and ethanol before antigen retrieval. Then, sections were permeabilized using PBS with 0.1% Triton and blocked with 3% BSA. Tunel Assay Kit (Servicebio, Cat# G1501) was used to detect DNA breaks formed during apoptosis. Perilipin-1 at 1:100 dilution (Cell Signaling, Cat# 9349S) was used to stain all adipocytes. DAPI was used to stain nuclei.

Tissues were homogenized in RIPA buffer with protease and phosphatase inhibitor cocktail. Proteins were separated using SDS-PAGE and transferred to PVDF membrane (Millipore) by iBlot 2 Western Blot Transfer System. Quantification of immunoblots was performed using ImageJ (NIH). Following antibodies were used: Vinculin (Abcam,#ab18508), Caspase1 (AdipGen Life Sciences, #AG-20B-0042); Caspase1(ThermoFisher, #PA5-38100; AdipGen Life Sciences, #AG-20b-0044); Caspase3 (Cell Signaling Technology, #9661); Caspase11 (Abcam, #ab180673); Syntaxin 4 (Synaptic Systems, #110042); Tubulin (Cell Signaling Technology, #2144); Hmgb1 (Abcam, #ab67281); UCP1 (Abcam, #ab10983); Nlrp1b (Novus Biologicals, #NBP1-54899); Cleaved Nlrp1b (AdipGen Life Sciences, #AG-20B-0084); PGAM1 (Cell Signaling Technology, # 12098); HSP70 (Abcam, #abab181606); AIM2 (Abcam, #ab93015); NLRP3 (AdipGen Life Sciences, #AG-20B-0006); GAPDH (Abcam, #ab8245); GM130 (Cell Signaling Technology, #12480); Caveolin (Cell Signaling Technology, # 3238); Myc (Cell Signaling Technology, # 2278); Flag (Sigma, #F1804); SNAP23 (Abcam, #ab3340); ATP5A (Abcam, #ab151229); TOM70 (Cell Signaling Technology, #65619); COXIV (Cell Signaling Technology, #4844); TOM20 (Cell Signaling Technology, #42406); OXPHOS (Abcam, #ab110413); Perilipin 1 (Cell Signaling Technology, #3470), F4/80 (Cell Signaling Technology, #70076).

Immunofluorescence staining was conducted according to standard procedures. Paraffin sections of tissue or cell climbing sheets were incubated with specific primary antibodies (GCA, PA5-77127, Invitrogen,1:200; F4/80, ab6640, abcam,1:200; PHB2, sc-133094, Santa Cruz,1:200; P65, CST8242S, Cell Signaling Technology,1:400, Ly6g/6c,Biolegend,108403,1:200;Perilipin-1,Cell Signaling Technology,9349 S,1:200) at 4 °C overnight. After washing with PBS for 3 times, the sections or sheets were incubated with corresponding fluorescent secondary antibodies. The cell nuclei were labeled with DAPI. The cytoplasmic membrane was stained with Dil. The results were imaged by fluorescence microscope or confocal microscopy.