Soluble recombinant human trail

All chemicals were purchased from Sigma Aldrich (St. Louis, MO, USA) unless otherwise specified. Soluble recombinant human TRAIL was obtained from Enzo Life Sciences (San Diego, CA, USA). The pan-caspase inhibitor Z-VAD-FMK was purchased from Merck Millipore (Darmstadt, Germany). All insoluble reagents were dissolved in dimethylsulfoxide (DMSO) and diluted with high glucose-containing DMEM supplemented with 10% fetal bovine serum (FBS) or Hank’s balanced salt solution (HBSS, pH 7.4; Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) (final DMSO concentration, <0.1%) before use.

Soluble recombinant human TRAIL and mdivi-1 were obtained from Enzo Life Sciences (San Diego, CA). Anti-human TRAIL-R4 (clone 104918 #MAB633) and anti-human TRAIL-R5 (clone 71903 #MAB631) were purchased from R&D systems (Minneapolis, MN). Mouse anti-Drp1 monoclonal antibody (C-5, #sc-271583), goat polyclonal anti-Fis1 antibody (K-14, sc-48865), and mouse monoclonal anti-Mfn1 antibody (D-10 sc-166644) were obtained from Santa Cruz Biotechnology (Dallas, TX).

Chemotherapeutics used for this study were cisplatin (Teva; Ulm, Germany), 5-fluorouracil (Medac; Hamburg, Germany) and gemcitabine (Hexal; Holzkirchen, Germany). Methylthiazolyldiphenyl-tetrazolium bromide (MTT) was obtained from Sigma Aldrich (St. Louis; MO, USA), human recombinant interferon beta from R&D System (New York; NY, USA), the PD-1 inhibitor nivolumab from Bristol-Myers (Anagni, Italy). Calcein-AM was purchased from Thermo Fisher (Eugene, USA), soluble, recombinant human TRAIL from Enzo Life Science (Paris, France). Primary mouse monoclonal antibody against PD-1, clone 913429 was obtained from R&D System (Wiesbaden, Germany), anti-human B7-H1/PD-L1 monoclonal antibody, clone 130021 from R&D System (Minneapolis, USA). Knock-down experiments were performed using PD-L1-siRNA (Dharmacon; Freiburg, Germany, Cat. Nr. L-015836-01-0010), RELA-siRNA for NF-κB (Dharmacon; Cat. Nr. L-003533-00-0005) and scrambled RNA (Dharmacon; Cat. Nr. D-001810-20-0005). Antibodies used for immunoblotting were mouse anti-human β-actin (Cell Signaling; Danvers, MA, USA), mouse anti-human-PD-1 (R&D System), mouse anti-human-PD-L1 (Clone # 130021; R&D System) and mouse anti-human-RELA/NF-κB p65 (Clone #532301; R&D System). The goat anti-mouse IgG secondary antibody was purchased from Santa Cruz Biotechnology (Heidelberg, Germany). NF-κB inhibitor—BMS-345541 was obtained from Sigma Aldrich.

All chemicals were purchased from Sigma Aldrich (St. Louis, MO, USA) unless otherwise specified. Soluble recombinant human TRAIL was obtained from Enzo Life Sciences (Farmingdale, NY, USA). The pan-caspase inhibitor Z-VAD-FMK was purchased from Merck Millipore (Darmstadt, Germany). All insoluble reagents were dissolved in dimethyl sulfoxide and diluted with high glucose-containing DMEM supplemented with 10% fetal bovine serum (FBS) or Hank’s balanced salt solution (pH 7.4, Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) (final dimethyl sulfoxide concentration, <0.1%) before use.