Hpdl1

Cells were plated at 2×10⁶ per P10 plate. Fresh media (6 ml) was added to the cells and collected after 24 hours. Collected media was spun at 4°C, 5 min, 1200 rpm to remove any cells and cell debris. After debris removal, the protein content in the media was concentrated 6 to 8-fold by using the 10K protein concentrator PES (Thermo Scientific) at 4°C. The protein concentrate was reconstituted in Cell Lysis Buffer (Cell Signaling Technology) and analyzed by Western blot.
Antibodies, Reagents, and Drugs. HIF1A, Lamin A/C, hPDL1, anti-biotin HRP-linked, and anti-rabbit IgG1 HRP-linked antibodies were purchased from Cell Signaling Technologies (Danvers, MA, USA); Actin, HuR 3A2, PDL1, donkey anti-goat IgG-HRP, goat anti-mouse IgG1-HRP antibodies were from Santa Cruz Biotechnology (Dallas, Texas, USA); the mPDL1/B7-H1 antibody was from B&D systems (Minneapolis, MN, USA); the alpha Tubulin antibody was from Sigma (Sigma-Aldrich). Mouse PDL1 ELISA Kit was purchased from Boster (Pleasanton, CA, USA). MLN4924 was purchased from Active Biochem (Kowloon Bay, Hong Kong); the PD1/PDL1-inhibitor-1 was purchased from Cayman Chemical (Ann Arbor, Michigan, USA).

Antigen retrieval was performed for tissues using 0.1 mol/L sodium citrate (pH 6.0) buffer or manufacturer's buffer. Sections were blocked using 3% H₂O₂, 4% fish skin solution (Electron Microscopy Sciences, Hatfield, PA) or goat‐serum (Sigma‐Aldrich, St. Louis, MO). Primary antibodies against h‐PD‐L1 (1:100) (Cell Signaling Technology, Danvers, MA), h‐Ki‐67 (1:100) (Neomarkers, Fremont, CA), cleaved caspase 3 (1:50) (Biocare Medical, Concord, CA), m‐F4/80 (1:200) and CD163 (1:100) (Abcam, Cambridge, MA), m‐NKp46 (1:50) (Biolegend, San Diego, CA), or CD68 (1:50) (BD Biosciences, San Jose, CA) were added overnight at 4°C. Anti‐rabbit‐IgG‐HRP (Santa Cruz Biotechnology, Dallas, TX) or anti‐rat‐IgG‐HRP (Jackson Immunoresearch, Westgrove, PA) secondary antibodies (1:1000) were used. 3, 3′‐diaminobenzidine (DAB) and hematoxylin counterstaining was performed followed by image capture (Leica Microsystems Inc., San Jose, CA) and quantification Simple PCI (Hamamatsu) 14.