Anti hif 1α

ChIP assay was performed as previously described [84 (link)]. Briefly, TSCC15 cells were cross-linked with 1% formaldehyde for 10min. ChIP assays were performed using anti-HIF1α (Novus, USA; BD, USA) and anti-DNMT1 (Abcam, UK). Anti-IgG was used as a negative control. The DNA fragments were extracted and subjected to qPCR by primers detecting for corresponding cis-acting elements and negative control regions.

Binding between the promoter region of Tie1 and HIF-1α was assessed used a ChIP assay kit (Millipore) following the manufacturer’s instructions. Briefly, A549 cells were incubated under hypoxic conditions for 24 h and then sonicated to obtain fragmented DNA. Chromatin was immunoprecipitated with anti-HIF-1α (Novus Biologicals, Littleton, CO, USA) or rabbit IgG (Sigma-Aldrich). The region in the Tie1 promoter containing the HIF-1α-binding site (5′-CTCGTG-3′, from − 1032 to − 1038 bp relative to the translation start site) was detected and amplified by PCR using the following primers: forward 5′-CATCCCAACCATTCCATTCCG-3′ and reverse 5′-TTCCCAGAACGGAACAAGACC-3′.

The canine CHMp-5b (5b) and CHMp-13a (13a) breast cancer cell lines [37 (link)] were kindly provided by Dr. Takayuki Nakagawa (The University of Tokyo, Tokyo, Japan) and cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS), 5 mg/L gentamicin sulfate and 6 mg/L fungizone. The canine ACE1 PCa cell line [38 (link), 39 (link)] was kindly provided by Dr. Tom Rosol (The Ohio State University, Columbus, OH) and cultured in RPMI-1640 medium containing 10% FBS, penicillin (100 U/ml) and streptomycin (100 μg/ml). Normal canine renal MDCK, normal human embryonic kidney HEK293 and human PC-3 PCa cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultured in RPMI-1640 medium containing 10% FBS, penicillin (100 U/ml) and streptomycin (100 μg/ml). 4′,6-diamidino-2-phenylindole (DAPI), cobalt chloride, rifampicin (RIF) and bromosulfophthalein (BSP) were purchased from Sigma-Aldrich (St. Louis, MO). Dimethyloxaloylglycine (DMOG) was purchased from Millipore (Billerica, MA). Rabbit polyclonal anti-HIF-1α and anti-OATP2B1 antibodies both predicted to react with canine species were purchased from Novus Biologicals (Littleton, CO).

Gefitinib, simvastatin, and fluorescent dyes were obtained from Melone Pharmaceutical (Dalian, China). Osimertinib was from Selleck Chemicals (Texas, USA), and murine cytokines were from Peprotech (New Jersey, USA), and LPS from Sigma-Aldrich (St. Louis, USA). Soybean phosphatidylcholine (SPC), cholesterol, and DSPE-PEG₂₀₀₀, DSPE-PEG-NHS, and DSPE-PEG-Mal were obtained from Advanced Vehicle Technology (Shanghai, China). The derivative T12 peptide (sequence: CGGGTHRPPMWSPVWP) was synthesized by Bankpeptide Biological Technology (Hefei, China). The primary antibodies of EGFR, phospho-EGFR (Tyr1068), Erk1/2, phosphor-Erk1/2 (Thr202/Tyr204), Akt, phosphor-Akt (Ser473), VEGFR2, VEGF, caspase3, cleaved-caspase3, LC3, TGF-β, GAPDH, and galectin-3 were purchased from Cell Signal Technology (Boston, USA). The primary antibody β-Actin was obtained from Sigma-Aldrich (St. Louis, USA). The primary antibodies of CD206 and TNF-α were from Abcam (Cambridge, UK). Anti-HIF-1α was obtained from Novus (Shanghai, China) and anti-IFN-γ was from Absin (Shanghai, China). The horseradish peroxidase (HRP)-conjugated goat anti-rabbit/mouse lgG secondary antibody was obtained from Beyotime (Shanghai, China). All other reagents (analytical grade) were provided by Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China).

Approximately 1 mg of total proteins was incubated overnight at 4°C with anti‐Sirt1 antibody (Cell Signaling) or anti‐HIF‐1α antibody (Novus Biologicals, Littleton, CO, USA) followed by precipitation with 20 µl of protein A/G‐Plus‐Agarose (Santa Cruz, Dallas, TX, USA) for 4 hr at 4°C. The precipitated complexes were immunoblotted with anti‐Sirt1 (Cell Signaling), anti‐HIF‐1α (Novus Biologicals), or anti‐acetyl‐lysine (Cell Signaling).