Rapamycin tlrl rap

AllStars negative control siRNA and Stealth RNAi for NR3C1 were from Qiagen. CellTiter-Glo® Luminescent Cell Viability Assay (G7570) and CellTiter 96® AQueous One Solution Cell Proliferation Assay (G3582) were purchased from Promega. Matrigel® Basement Membrane Matrix, Growth Factor Reduced (GFR) was purchased from Corning. Antibodies against LRP5 (5731s), LRP6 (3395s), p-β-catenin (4176s), Cyclin D1 (2978s), LC3A/B (4108s) and p62 (5114 s) were purchased from Cell Signaling Technology. Antibodies against α-smooth muscle Actin (α-SMA) (ab5694), CD68 (ab53444), GAPDH (ab8245) and recombinant mouse Wnt3a protein (ab81484) were purchased from Abcam. Antibodies against CD31 (553370) and CD102 (553326) were purchased from BD Bioscience. Anti-GR (sc-393232) was purchased from Santa Cruz Biotechnology. Recombinant mouse sFRP3 protein (592-FR/CF) was purchased from Bio-techne. Rapamycin (tlrl-rap) was purchased from InvivoGen. Premo™ Autophagy Tandem Sensor RFP-GFP-LC3B Kit including chloroquine (P36239) was purchased from Thermo Fisher Scientific.

Dopamine hydrochloride (#H-8502), S-(-)3-PPP (#P-103), Terguride (#T-134), angiotensin II (#A9525), and buprenorphine hydrochloride (#B9275) were purchased from Sigma-Aldrich; sulpiride (#0895), quinpirole (#1061), bromocriptine (#0427), olmesartan (#4616), Dynorphin B (#3196), naloxone hydrochloride (#0599), WN552122 (#1038), and rimonabant hydrochloride (#0923) from Tocris; DAMGO (#024-10) from Phoenix Pharmaceuticals, and rapamycin (#tlrl-rap) from Invivogen and morphine sulfate (#M1167) from Spectrum Chemicals.

KOD-plus- (KOD-201) was employed for high-fidelity polymerase chain reactions (TOYOBO). Huh7.5.1 cells derived from the Huh7 cell line (ATCC CCL-185) were cultured in a Dulbecco's modified Eagle Medium (DMEM; Wako, 045-30285) containing 10% fetal calf serum (JRH biosciences/SIGMA, 12603C) and 1% nonessential amino acids (Invitrogen, 11140050). Polyclonal antibodies against human LC3 was described previously [10] (link). The mouse monoclonal antibody, clone M2, against FLAG peptide (DYKDDDDK) (F1804), ammonium chloride (254134), and chloroquine diphosphate salt (C6628) were purchased from SIGMA-ALDRICH, the mouse monoclonal antibodies against GAPDH (ab8245) were from Abcam, and the rabbit monoclonal antibody against Myc epitope tag (2278) was from Cell Signaling Technology. Protein concentrations were determined using the bicinchoninic acid protein assay reagent (Pierce, 23225). E64d (4321-v) and pepstatin A (4397-v) were purchased from Peptide Institute, and the rapamycin (#tlrl-rap) was from InvivoGen. Torin1 was a kind gift from Dr. Nathanael S. Gray at the Dana Farber Cancer Institute and Dr. David Sabatini at the Whitehead Institute for Biomedical Research. To introduce the plasmid into the cells, FuGENE HD transfection reagent (E2311) was used (Promega).