Ab23673

Gomisin N (≥98% purity, C₂₃H₂₈O_6, GN) was purchased from YuanYe Biotechnology (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM), bovine calf serum (BCS), fetal bovine serum (FBS), penicillin–streptomycin (P/S), recombinant human BMP4, trypsin and ethylenediaminetetraacetic acid (EDTA) were purchased from Gibco (Gaithersburg, MD, USA). Dexamethasone (DEX), 3-isobutyl-1-methylxanthine (IBMX), insulin, Oil Red O (ORO), dimethyl sulfoxide (DMSO), phosphatase inhibitor cocktails I and II, AICAR and CC were purchased from Sigma-Aldrich (St. Louis, MO, USA). Phosphate-buffered saline (PBS) was purchased from iNtRON Biotechnology (Gyeonggi, South Korea). Antibodies against C/EBPα (sc-61), FAS (sc-20140), SREBP1c (sc-366), LPAATθ (sc-514164), DGAT1 (sc-32861), Lipin1 (sc-98450), PPARα (sc9000), PPARγ (sc7196), PGC1α (sc13067), CPT1 (sc393070), UCP1 (sc6529) and GAPDH (sc365062) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Antibodies against p-ACC (cs3661, Ser79) p-AMPK (cs2535, Thr172), AMPK (cs2603), p-HSL(cs4139) and ACC (cs3662) were purchased from Cell Signaling Technologies (Danvers, MA, USA). Antibodies against PPARδ (ab23673), PRDM16 (ab202344), UCP1 (ab23841), FGF21 (ab64857) and CPT1 (ab1285568) were purchased from Abcam (Cambridge, UK).

Total protein was extracted from the ALT astrocytes and spinal cord tissues using a lysis buffer (20 mM Tris-HCl, 0.1% sodium dodecyl sulfate (SDS), 0.8% NaCl, and 1% Triton X-100). The protein was subjected to gradient electrophoresis on a 12% gel. The resolved proteins were electroblotted onto a nitrocellulose membrane. The membrane was incubated with a blocking reagent. Next, the membrane was incubated with the following primary antibodies at 4°C for 12 h: anti-IκB alpha (E130) (1 : 2000; ab32518; Abcam, Cambridge, MA, USA), anti-PPAR-β (1 : 2000; ab23673; Abcam), anti-GFAP (1 : 2000; ab7260; Abcam), anti-β-actin (1 : 4000; ab8227; Abcam), and anti-CISD2 (1 : 500; PA5-34545; Thermo Fisher Scientific). The membrane was washed and incubated with goat anti-rabbit IgG (horseradish peroxidase- (HRP-) conjugated secondary antibody) (1 : 5000; 12-348; Merck Millipore) for 1 h. Protein bands were developed using the Immobilon™ Western Chemiluminescent HRP Substrate (WBKLS0500; Merck Millipore). Densitometric analysis of the protein bands was performed using ImageQuant™ LAS 4000 (GE Healthcare Life Sciences).

HSCs were co-cultured with either SFM, hBM-MSCs, or hBM-MSCs-Ex (5 ng/ml) for 48 h before samples were collected for protein extraction. Liver tissue was collected from each treatment group (liver fibrosis, hBM-MSCs, and hBM-MSCs-Ex group) for protein extraction. Protein samples were mixed with SDS sample buffer and heated to 95 °C for 10 min, followed by separation on SDS-polyacrylamide gels. Resolved proteins were electro-blotted onto nitrocellulose membrane and probed with antibodies against PPARγ (ab 23673), Wnt3a (ab 248472), Wnt10b (ab70816), β-catenin (ab32572), WISP1 (ab50041), Cyclin D1 (ab16663), α-SMA (ab5694), Collagen I (ab138492), and GAPDH (ab 8245) overnight at 4 °C (1:1000 dilution, Abcam, Cambridge, UK). Nitrocellulose membranes were then incubated with a secondary antibody, HRP-conjugated goat anti-rabbit IgG (ab15007), at room temperature for 2 h, and visualized by chemiluminescent detection according to the manufacturer’s instructions (Immobilon western chemiluminescent HRP substrate, Millipore).

Tissues and cells were lysed in RIPA buffer according to manufacturer’s instruction (Beyotime, China). Protein lysates were heated at 95°C 5min in 5 × Sodium Dodecyl Sulfate (SDS) sample buffer separated by 12% SDS-PAGE (20 μg each lane), then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA) using Mini Trans-Blot Cell (Bio-Rad, USA). After being blocked with 5% non-fat milk for 1.5 h, the membranes were incubated with primary antibodies overnight at 4°C. After washed three times, the membranes were hybridized with secondary antibody for 1 h at 37°C, and washed three times. The targeted proteins were detected using the ImageQuant LAS 4000 mini (GE, USA) according to the manufacturer instructions. Primary antibodies were specific for PPARD (Abcam, USA, ab23673; 1:1000 dilution), TCF7L2 (Abcam, USA, ab32873; 1:1000 dilution), and β-actin (Boster, China, BM0627; 1:1000 dilution). Secondary antibodies were goat anti-mouse IgG-HRP (Santa Cruz, USA, sc-2005; 1:3000 dilution) and goat anti-rabbit IgG-HRP (Santa Cruz, USA, sc-2004; 1:3000 dilution).