Rapamycin

DRibbles were enriched from the cells described above as published previously (6 (link)). The tumor cells were pretreated with 100 nM Bortezomib (Millennium pharmaceuticals, Cambridge, MA), 100 nM Rapamycin (Enzo Life Sciences, Shanghai, China) and 10 mM NH₄Cl (Enzo Life Sciences, Shanghai, China) for 18–24 h in a 5% CO2 incubator at 37°C. The cells were harvested and centrifuged at 1,000 rpm for 10 min to remove cells and large cell debris, then the supernatant was centrifuged to pellet the DRibbles at 12,000 rpm for 30 min. The DRibbles were washed three times with PBS and Bicinchoninic acid Protein Assay kit (Beyotime Biotechnology, Shanghai, China) was used to quantify the total protein concentration in DRibbles. In some experiments, the tumor cells were pretreated with Rapamycin (100 nM) alone to isolate the autophagosomes.

HepG2.2.15 cells containing transfected HBV full-length DNA [17 (link)] or HepG2 cells were treated with 200 nmol/L Bortizomib (Millennium pharmaceuticals, USA) alone or in combination with 100 nmol/L Rapamycin (Enzo Life Sciences, China) or 30 mmol/L NH₄Cl for 18 h, DRibbles containing autophagosomes were prepared from the culture media as described [13 (link),14 (link)]. Morphology analysis of HBV⁺ DRibbles was done under transmission electron microscopy. HBsAg in HepG2.2.15 cell lysates was detected by western blot analysis using anti-HBsAg antibody (Santa Cruz, Biotechnology, Inc., USA). LC3 in both HepG2.2.15 cell lysates and HBV⁺ DRibbles was determined by western blot analysis using the polyclonal LC3B antibody (Cell Signal Technology, USA,1:1000), the HRP-labeled goat anti-rabbit IgG secondary antibody (1:5000) and a chemiluminescence kit (Multisciences Biotech Co., Ltd., China). Levels of HBsAg and HBeAg in HBV⁺ DRibbles were measured by ELISA (Shanghai Kehua Bioengineering Co., Ltd., China).

Anticancer drug cisplatin was obtained from Sigma–Aldrich. cisplatin 1mM stock was prepared in normal saline (0.9% NaCl) and stored as aliquots at -20°C up to 3 months. V-ATPase inhibitor bafilomycin A (Sigma–Aldrich, M17931) was dissolved in DMSO at a 100μM stock solution. Autophagy modulators rapamycin and chloroquine were procured from Enzo Life Sciences, USA. rapamycin was dissolved in DMSO at a 500μM stock solution. chloroquine was dissolved in deionized water for a 60 mM stock solution. For long term storage, all stock solutions were stored at -20°C. Selective MEK inhibitor cobimetinib (10mM in DMSO) was obtained from ApexBio and the stock solutions were stored at -20°C.

The human epidermoid carcinoma A431 cell line, which overexpresses CCKBR, was generated and kindly provided by Dr. Luigi Aloj 23 (link). The A431 and A431/CCKBR cell lines were cultured in DMEM, whereas the rat pancreatic acinar AR42J cells (ECACC, UK) were grown in RPMI medium, supplemented with 10% FCS, 2 mM glutamine and antibiotics (0.1 mg/mL streptomycin, 100 IU penicillin) at 37 °C and 5% CO₂. For CCKBR-specific knock-down, duplex siRNAs against CCKBR or control duplex against luciferase (Microsynth) were used at a final concentration of 100 nM in Optimem (Gibco): CCKBRseq1 sense RNA 5′-UAUACGAGUAGUAGCACCAdTdT-3′, CCKBRseq2 sense RNA 5′-CCGCCAAAGGAUGGAGUACdTdT-3′ and control sense RNA 5′-CGUACGCGGAAUACUUCGAdTdT-3′. Cells at 60-80% confluence were transfected with siRNAs by using Lipofectamin 3000 according to the manufacturer's recommendations. RAD001 (Selleckchem), rapamycin (Enzo Life Sciences), BML-257(Enzo), SC-514 (Enzo) or metformin (Selleckchem) were diluted in DMSO or water, respectively.

Recombinant human TGF-β, recombinant FGF-2 and recombinant human TNF-α were purchased from Biolegend (San Diego, CA). Rapamycin was from ENZO life science (Loerrach, Germany). Bafilolycin A1 was from Invivogen. FITC-PHA was from Vector (FL-1111). FITC-PNA was from Sigma (L7381). DAPI was from Sigma (D9542). Alexa Flour 488, 594 or 647 conjugated anti-mouse or anti-rabbit IgG were from Jackson ImmunoResearch Laboratories (West Grove, PA). The primary antibodies against JLP (ab12331, 1:1000), Fsp-1 (ab197896, 1:200), α-SMA (ab124964, 1:1000), Fibronectin (ab45688, 1:500), Collagen-I (ab34710, 1:1000), Ki67 (ab16667, 1:250), TGF-β (ab92486, 1:500), phospho-Smad2 (ab188334, 1:1000), phospho-Smad3 (ab52903, 1:1000) and p62 (ab56416, 1:200) were all from Abcam (Cambridge, MA). Anti-LC3 antibody (ab51520, 1:100, Abcam) was used for immuno-staining and Anti-LC3 antibody (L7543, 1:1000, Sigma) was used for western blotting, respectively. Anti-Nephrin and anti-F4/80 (123120, 1:100) antibodies were from Progen and Biolegend, respectively. Anti-Caspase-3 (#9664, 1:1000), anti-Beclin 1 (#3738,1:1000) was from Cell signaling Technology (Danvers, MA). Anti-GAPDH (sc-365062, 1:2500) was purchased from Santa Cruz (Santa Cruz, CA).