Cathepsin b

The immunoblotting experiment was performed as previously described [19 (link),20 (link)]. In brief, total protein was isolated from tissue samples using RIPA lysis buffer with protease inhibitor cocktail tablets (Roche, Switzerland), and quantified using a BCA Protein Assay Kit (Thermo Fisher Scientific, U.S.A.). The total protein samples were loaded and separated on TGX Stain-Free™ FastCast™ Acrylamide Kit (Bio-Rad, U.S.A.) and transferred to PVDF membranes (Merck Millipore, Germany). The membranes were blocked with 5% skim milk for 2 h and incubated with primary antibodies against cathepsin D (Abcam, U.S.A., 1:2000), Bax (ProteinTech, China, 1:200), cathepsin B and Bcl-2 (Cell Signaling Technology, U.S.A., 1:1000) overnight at 4°C, which was followed by incubation with the corresponding secondary antibodies for 2 h at room temperature. Signals were visualized by enhanced chemiluminescence (ECL) reagents (Abvansta, U.S.A.) and captured by a Chemi Doc^MP Imaging System (Bio-Rad, U.S.A.). Total protein was used for normalization. Immunoreactive bands were quantified using ImageJ.

Caerulein (CER) (#HY-A0190) was purchased from MedChemExpress (Monmouth Junction, NJ). Palmitoleic acid (POA), CCK, L-Arginine (L-Arg), and Na taurocholate (NaT) were purchased from Sigma-Aldrich Chemical (St. Louis, MO, USA). BSA was purchased from Yeasen (Shanghai, China). JQ1, EX527 were purchased from Selleck Chemicals (Houston, TX, USA). Antibodies against microtubule associated protein 1 light chain 3 beta (LC3B) (#3868S), p62 (#23214S), ATG14 (#96752S), cathepsin B (#31718S), AMP activated protein kinase (AMPK) (#5831T), phosphorylated AMPK (#2535T), mammalian target of rapamycin (mTOR) (#2983T), phosphorylated mTOR (#5536T), β-actin (#3700s) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibody against Amylase (#sc46657) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies against BRD4 (#ab128874), LAMP2 (#ab203224), syntaxin 17 (STX17) (#ab229646), cathepsin L (#ab6314) were purchased from Abcam (Cambridge, MA, USA). Antibody against CD45 (#550539) were purchased from BD Biosciences (Franklin, NJ, USA).

Melanoma cells were cultured in six-well dishes then transfected with indicated siRNA. 72 h after transfection, cells were lysed in buffer containing 50 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 1% Triton X-100, protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). Samples were dosed using Pierce BCA Protein Assay kit (ThermoFisher) and 30 µg of total extracts were resolved by 10% SDS-PAGE, transferred to nitrocellulose membranes, and then exposed to the appropriate antibodies: anti-βactin-HRP (Sigma-Aldrich A3854, diluted at 1: 5000), Vinculin (Cell Signaling Technology 13901 S, diluted at 1:1000), TFEB (Cell Signaling Technology 4240 S, diluted at 1:1000), LC3A/B (Cell Signaling Technology 4108, diluted at 1:1000), IGF2R (Cell Signaling Technology 14364 S, diluted at 1:1000), Cathepsin B (Cell Signaling Technology 31718, diluted at 1:1000), TFE3 (Cell Signaling Technology 14779 S, diluted at 1:1000), Phospho-pSMAD2 (Cell Signaling Technology 18338, diluted at 1:1000), SMAD2 (Cell Signaling Technology 5339, diluted at 1:1000), laminA/C (Cell Signaling Technology 2032, diluted at 1:1000), Phospho-4E-BP1 (Ser65) (Cell Signaling Technology 9451, diluted at 1:1000), 4E-BP1 (Cell Signaling Technology 9644, diluted at 1:1000).
Proteins were visualized with the ECL system (GE Healthcare) using horseradish peroxidase conjugated secondary antibodies.