Phospho ampkα thr172

Total protein from tissues or cells was prepared with RIPA lysis buffer supplemented with protease and phosphatase inhibitors (Roche). The protein samples were boiled for 10 min and then were separated by 10% sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (Millipore). Next, the membranes were blocked with 5% milk for 1 h at room temperature and incubated with the corresponding antibodies against UCP1 (Abcam), PGC-1a (Millipore), β-Actin (Cell Signaling Technology), AMPK (Cell Signaling Technology),Phospho-AMPKα (Thr172) (Cell Signaling Technology), p38 MAPK (D13E1)XP® (Cell Signaling Technology),Phospho-p38 MAPK (Thr180/Tyr182) (Cell Signaling Technology), Phospho- (Ser/Thr) PKA Substrate (Cell Signaling Technology), Tubulin (Sigma) and IRF4 (Santa Cruz) overnight at 4 °C. Subsequently, the membranes were incubated with the secondary antibody for 1 h at room temperature. An ECL detection system was used to detect the signals (GE Healthcare, USA).

The MOSEC line was a kind gift from Dr. Honami Naora (The University of Texas MD Anderson Cancer Center). Stable MOSEC lines were generated as previously described [41 (link)]. The MOSEC lines were cultured in DMEM medium [42 (link)]. The human ovarian cancer cell line SKOV3 and ovarian clear cell carcinoma cell line ES-2 were provided by Dr. Gordon Mills (The University of Texas MD Anderson Cancer Center) and Dr. Patrice Morin (National Institute on Aging, Baltimore, Maryland, USA), respectively and were cultured in McCoy’s 5A medium. All cell culture reagents used were obtained from Invitrogen (Thermo Fisher Scientific Inc., Waltham, MA, USA). Metformin (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in DMEM containing 10% fetal bovine serum (FBS) at the indicated concentration. Carboplatin (Sigma-Aldrich) was dissolved in water and diluted in DMEM to various concentrations. Primary antibodies against AMPKα, phospho-AMPKα (Thr172), AKT, phospho-AKT (Ser473), mTOR, phospho-mTOR (Ser2448) S6, phospho-S6 (Ser235–236), 4EBP1, phospho-4EBP1 (Thr37/46) and β-actin were obtained from Cell Signaling Technology (Danvers, MA, USA). All other chemicals were purchased from Sigma-Aldrich.

Total protein was prepared from cultured cells or mouse adipose tissues, and western blotting was performed as briefly described below. Proteins were quantified with a Pierce BCA Protein Assay Kit, separated by SDS-PAGE, and transferred to nitrocellulose membranes. Membranes were blocked with 5% nonfat milk dissolved in Tris-buffered saline with Tween 20 (TBST) at 37°C for 1 h and incubated with primary antibodies against AMP-activated protein kinase alpha (AMPKα) (Cell Signaling Technology, Cat# 2532, 1 : 1000), phospho-AMPKα (Thr172) (Cell Signaling Technology, Cat# 2531, 1 : 1000), β-actin (Santa Cruz, Cat# sc-47778, 1 : 1000), or GAPDH (Santa Cruz, Cat# sc-32233, 1 : 1000) at 4°C overnight. After incubation with horseradish peroxidase-conjugated secondary antibodies at 37°C for 1 h, proteins were detected using Pierce ECL Western Blotting Substrate and quantified using Quantity One 4.4.0 software (Bio-Rad, Hercules, CA, USA).

Western blot analyses were performed as previously described (Jiang et al., 2016 (link); Jiang et al., 2017a ; Jiang et al., 2017b (link)). The HDAC Antibody Sampler Kit (#9928), Phospho-HDAC4^Ser246/HDAC5^Ser259/HDAC7^Ser155 (#3443), HDAC2 (#5113), HDAC6 (#7558), H3K4me2 (#9725), H3K4me3 (#9727), H3K27me2 (#9728), H3K27me3 (#9733), Phospho-AKT^Ser473 (#4060), AKT (#4685), Phospho-mTOR^Ser2448 (#5536), mTOR (#2983), Phospho-AMPKα^Thr172 (#2535), AMPKα (#5831), Phospho-MEK1/2^Ser217/221 (#9154), Phospho-ERK1/2^{Thr202/Tyr204} (#4370), ERK1/2 (#4695), Phospho-JNK1/2 ^{Thr183/Tyr185} (#4668), JNK1/2 (#9258), Phospho-p38^{Thr180/Tyr182} (#4511), p38 (#8690), Phospho-β-Catenin^Ser675 (#4176), Non-phospho-β-Catenin^{Ser33/37/Thr41} (#8814), β-Catenin (#8480), Phospho-IKKα/β^Ser176/180 (#2697), IKKβ (#8943), Phospho-NF-κB p65^Ser536 (#3033), NF-κB p65 (#8242), Phospho-Smad2^Ser465/467 (#3108), Smad2 (#5339), Phospho-Smad3^Ser423/425 (#9520), Smad3 (#9523), Phospho-Smad1/5^Ser463/465 (#9516), and Smad1 (#6944) antibodies were obtained from Cell Signaling Technology. Antibodies against Histone H3 (ab1791), H3K36me2 (ab9049), H3K36me3 (ab9050), H4K20me3 (ab9053), H3K9me1 (ab9045), H3K9me2 (ab1220), H3K4me1 (ab8895), H3K23me1 (ab176132), H4K5ac (ab51997), H4K8ac (ab15823), H3K18ac (ab40888), H3K23ac (ab61234) and H4K12ac (ab46983) were purchased from Abcam.

After lysed with RIPA buffer (Solarbio, R0010), cell lysates were separated on SDS-PAGE gels and blotted onto membranes, then blocked with 5% milk for 1 h at RT and immunoblotted with primary antibodies at 4 °C overnight: γ-H2AX (Abcam, ab26350, 1:1000 dilution), caspase3 (Cell Signaling Technology, 9662, 1:1000 dilution), cleaved caspase3 (Cell Signaling Technology, 9664, 1:1000 dilution), β-actin (Fude Bio, FD0060, 1:5000 dilution), SLC7A11/xCT (Cell Signaling Technology, 126915, 1:1000 dilution), GPX4 (Abcam, ab125066, 1:1000 dilution), SCD1 (Abcam, ab236868, 1:1000 dilution), ACSL3 (Santa Cruz, sc-166374, 1:200 dilution), and Phospho-AMPKα (Thr172) (Cell Signaling Technology, 2535, 1:1000 dilution). Finally, the membranes were incubated with secondary antibodies conjugated to HRP (Fude Bio, 1:5000 dilution) and visualized by imaging systems.

After being treated for the desired period of time, HCT116 cells were collected and washed twice with phosphate-buffered saline and lysed in RIPA buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 1 mM phenylmethlsulfonyl fluoride, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM sodium vanadate, 1 µg/ml leupeptin). Equal amounts of protein were loaded and the immunoblotting was detected by Li-Cor Odyssey image reader as described^{32 (link)}. Anti-β-actin antibody was obtained from Santa Cruz Biotechnology. Antibodies against LC3-I/II, phospho-AMPKα (Thr172), ACC, phospho-ACC (Ser79), and PERK were obtained from Cell Signaling Technology. Anti-Sesn2 antibody was obtained from Proteintech.