P mtor 5536

GPR30 agonist G1 (10,008,933) was purchased from Cayman Chemical (Ann Arbor, MI, USA). GAPDH was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The rabbit anti-goat, goat anti-rabbit, and goat anti-mouse secondary antibodies were purchased from Zhongshan Company (Beijing, China). DAPI was obtained from Roche Molecular Biochemicals (Bayer, Mannheim, Germany). The ECL reagent was purchased from Millipore (Billerica, MA, USA). Anti-LC3B antibody(ab192890), F-actin(ab205), anti-BCL-2 antibody (ab182858), anti-BAX antibody(ab32503), and abti-P62 antibody (ab109012) were purchased from Abcam (Cambridge, MA, USA); AKT(9272), p-AKT(4060), mTOR(2983) and p-mTOR(5536) were purchased from Cell Signaling Technology (Beverly, MA, USA); AngII, Ad-mCherry-GFP-LC3 and rapamycin were purchased from Hanheng Biotechnology Co., Ltd. (Shanghai, China).

Acetonitrile, methanol (HPLC grade), and dichloromethane were purchased from Sinopharm Chemical Reagent Company (Shanghai, China). Formic acid (HPLC grade) was purchased from Dima Technology (Lake Forest, CA, USA). And the standard chemicals for metabolite identification were obtained from Sigma-Aldrich (St. Louis, MO, USA) and Aladdin Industrial Corporation (Shanghai, China). Distilled water was filtered through a Milli-Q system (Millipore Corporation, Billerica, MA, USA). CQ (C6628), AOM (A5486), and EBSS (E2888) were purchased from Sigma-Aldrich. DSS (36–50 kDa, 0216011080) was purchased from MP Biomedicals (Santa Ana, CA, USA). SNX10 (sc-104657) and GAPDH (sc-47724) antibodies were the product of Santa Cruz Biotechnology (Dallas, TX, USA). Antibody against LAMP-2A (ab18528) was purchased from Abcam (Cambridge, UK). Antibody against β-actin (AA128) was from Beyotime Institute of Biotechnology (Haimeng, China). mTOR (2983) and p-mTOR (5536) antibodies were the product of Cell Signaling Technology (Boston, MA, USA).

Apigenin (C15H10O5; purity ≥98%), hematoxylin–eosin (HE) staining kit, and Safranin O-fast green (SO) staining kit were purchased from Beijing Solarbio Science & Technology (Beijing, China); tert-butyl hydroperoxide solution (TBHP), chloroquine (CQ), and type II collagenase were obtained from Sigma-Aldrich (St. Louis, MO, United States); dorsomorphin (compound C) and bafilomycin A1 were purchased from MCE (Monmouth Junction, NJ, United States); the primary antibodies against Bcl2 (12789-1-AP), Bax (50599-2-Ig), MMP13 (18165-1-AP), β-actin (20536-1-AP), and TFEB (13372-1-AP) were obtained from Proteintech (Wuhan, China); p21 (ab109199), p62 (ab109012), collagen II (ab188570), ADAMTS5 (ab41037), AMPK (ab32047), p-AMPK (ab13448), LAMP2 (ab125068), and lamin B (ab133741) were acquired from Abcam; LC3 (#83506), cleaved caspase 3 (#9661), p-mTOR (#5536), and mTOR (#2972) and apigenin were partially purchased from Cell Signaling Technology (Danvers, MA, United States); p16INK4a (A0262), aggrecan (A11691), and LAMP1 (A2582) were purchased from ABclonal Technology (Wuhan, China). Goat anti-rabbit and anti-mouse IgG-HRP antibodies were purchased from Bioworld (Nanjing, China). Alexa Fluor® 594- and 488-conjugated secondary antibodies were obtained from Jackson ImmunoResearch (PA, United States); 4′,6-diamidino-2-phenylindole (DAPI) was purchased from Beyotime (Shanghai, China).

[2-(Methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl) ammonium hydroxide (SBMA), 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone (98%), and poly (ethylene glycol) dimethacrylate (PEGDMA, MW = 550 Da) were obtained from Sigma-Aldrich (MO, USA). Poly (ethylene glycol) methyl ether methacrylate (PEG, MW = 475 Da) was obtained from Aladdin (Shanghai, China). A hematoxylin–eosin (H&E) stain kit, BCA assay kit, and Masson’s trichrome (MTS) stain kit were purchased from Beyotime (Beijing, China). Collagen I (ab21286), collagen III (ab7778), laminin (ab11575), fibronectin (ab268020), CD31 (ab28364), VEGF (ab39256), CD68 (ab955), CD163 (ab182422), sQSTM1/p62 (ab56416), TNF-α (ab9755), goat anti-rabbit IgG Alexa Fluor® 647 (ab150083), and goat anti-mouse IgG Alexa Fluor 488® (ab150113) were obtained from Abcam (CA, UK). Matrix metallopeptidase2 (MMP-2) (384993) and laminin (383353) were obtained from Zenbio (Chengdu, China). Microtubule-associated one protein light chain 3 (LC3, L7543) was purchased from Sigma-Aldrich. PI3K (4228), p-PI3K (4257), AKT (4691), p-AKT (4060), mTOR (2983), and p-mTOR (5536) were obtained from Cell Signaling Technologies (Beverly, MA, USA). The RIPA lysis buffer was purchased from GE Healthcare Biosciences (PA, USA). The DAB Chromogen Kit was purchased from ZSGB-BIO (Beijing, China).

Mouse pancreatic tissues and cell lysates were homogenized in RIPA buffer (Cell Signaling, Danvers, MA) containing a protease inhibitor (Sigma, St Louis, MO). Anti-DC-SIGN (BS70885, Bioworld Technology), anti-mTOR (2972, Cell Signaling Technology), anti-phosphorylated mTOR (P-mTOR; 5536, Cell Signaling Technology) and anti-Myc (ab32072, Abcam) antibodies were used. ImageJ was used to analyze densitometric values on two different scans after background subtraction, from at least three different experiments.
Mouse serum was collected to measure the levels of lipase, amylase and cytokines. Enzyme-linked immunosorbent assays (ELISAs) were performed to measure cytokine levels in animal serum and cell supernatants according to the manufacturer’s instructions.

Western blots were performed as previously described [9 (link), 10 (link)] using the primary antibodies against MKL1 (ab14984) from Abcam, ERα (sc-543), and p-ERK (sc-7383) from Santa Cruz Biotechnology, ERK 1/2 (4695) from Cell signaling technology, and p-mTOR (5536) from Cell Signaling Technology.