Hif 1α

Total protein was extracted from the lysed CD8+ T cells. Protein concentration was determined using the Pierce™ Rapid Gold BCA Protein Assay Kit (Thermo Scientific, USA). Proteins were separated by 10% SDS-PAGE (Bio-Rad, USA) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). Membranes were blocked in 5% fat-free dried milk in phosphate-buffered saline containing 0.1% Tween-20 (PBST) solution and incubated with antibodies against HIF-1α (1 : 300; Proteintech, USA), PIK3CB (1 : 1000; Abcam, UK), and F2RL1 (1,1000; Abcam, UK). Band intensity was detected by using the ImageQuant™ LAS 4000 mini (GE Healthcare, USA).

Western blot assay was performed as detailed in the previous article [31 (link)]. Antibody details were as follows: VHL (#68547, Cell Signaling Technology), FBXO22 (13606-1-AP, Proteintech), HIF-1α (11587-1-AP, Proteintech), GAPDH (60004-1-AP, Proteintech), VEGF (19003-1-AP, Proteintech), Cyclin D1 (2978T, Cell Signaling Technology), Cyclin E2 (4132T, Cell Signaling Technology), Flag tag (66008-4-Ig, Proteintech), HA tag (51064-2-AP, Proteintech), Ubiquitin (#3936, Cell Signaling Technology. All Western blot experiments were repeated a minimum of three times.

A monoclonal antibody against human hypoxia-inducible factor-1α (HIF-1α; 1:200; Proteintech, Wuhan, China) and a monoclonal antibody against rabbit matrix metalloproteinase-9 (MMP-9;1:500; Servicebio, Wuhan, China) were performed as the primary antibodies in the present study, respectively. As for secondary antibodies, the horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse immunoglobulin G antibody (1:200; Servicebio, Wuhan, China) were used.

Treated cells were collected by adding RIPA buffer (Beyotime, Shanghai, China), phenylmethylsulfonyl fluoride (Beyotime, Shanghai, China), and phosphatase inhibitor (Beyotime, Shanghai, China) for 30 min, and the supernatant was collected by centrifuging the lysate (12,000 rpm, 15 min). Protein samples were separated by 10% sodium dodecyl-sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes, which were then closed at room temperature for 10 min with Rapid Closure Solution. The membranes were then incubated for 12 h using appropriate primary antibodies against the following proteins: inducible nitric oxide synthase (iNOS; 1:1,000; Cell Signaling Technology, Boston, MA, USA); CD80 (1:2,000; Proteintech, Wuhan, China); GYS1 (1:5,000; Abcam, Cambridge, MA, USA); HIF-1α (1:1,000; Proteintech, Wuhan, China); P2Y₁₄ (1:1,000; ABclonal, Wuhan, China) and β-actin (1:2,000; Proteintech, Wuhan, China). Next, secondary antibody:HRP-labeled goat anti-rabbit immunoglobulin G (1:1,000; Beyotime, Shanghai, China) was used at room temperature for 1 h. Protein signals were captured with a Gel Document System (SYNGENE, Cambridge, UK), and quantitative analysis of the protein was performed using ImageJ software.

Proteins were extracted from tissues or cells in RIPA buffer (NaCl 150 mM, Nonidet P-40 1%, sodium deoxycholate 0.5%, SDS 0.1%, Tris 25 mM, PH = 7.4) containing protease inhibitor cocktail (Merck). PVDF membrane was probed with primary antibodies at 4 °C overnight and incubated with HRP-conjugated secondary antibodies in room temperature for 1 hr. The protein bands were visualized by ECL Western Blotting Substrates (Bio-Rad). Antibodies used are: PDH2 (1:1000, Cell Signaling Technology, #4835 S, D31E11), iNOS (1:1000, Thermo Fisher Scientific, #PA3-030A), HIF-1α (1:1000, Proteintech, #20960-1-AP), HIF1α-OH-564 (1:1000, Cell Signaling Technology, #3434 S, D43B5), LDHA (1:1000, Cell Signaling Technology, #2012S), β-Actin (1:1000, Cell Signaling Technology, #4970 S, 13E5), His-tag (1:1000, R&D Systems, #MAB050R, AD1.1.10), rabbit IgG (1:2000, Cell Signaling Technology, #7074P2), mouse IgG light chain (1:2500, Cell Signaling Technology, #58802 S, D3V2A).

Protein in cell lysates were separated by SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Millipore, Hessen state, Germany), followed by incubated with primary antibodies against CLEC3B (Abcam, Massachusetts, US), HIF-1α, VEGF (Proteintech, Chicago, US), phospho-AMPKα, AMPKα, CD31, CD34 and EMT associated molecules (Cell Signaling Technology, Danvers, US) overnight in 4 °C. Next day, the membranes were subsequently incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz Biotechnology, California, US). The immunoreactive protein were visualized by using enhanced chemiluminescence detection kit (Tiangen Biotech, Beijing, China) and image analyzer ImageQuant LAS 4000 (GE Healthcare, Abingdon, UK). To ensure equal loading of plasma protein, the membranes were stained with 0.2% Ponceau S (Sinopharm Chemical Reagent, Shanghai, China) to ensure equal loading of the proteins.

Skin tissue was fixed overnight in 4% paraformaldehyde (PFA) and embedded in paraffin. Paraffin sections were cut at a 4 μm thickness. IHC was conducted by using the Opal™ 7-color manual kit (PerkinElmer, USA) according to the manufacturer's protocol. Antigen retrieval was performed in citrate buffer (pH 6.0) using a high-pressure method. Primary antibodies were incubated for 1 h in a humidified chamber at room temperature (RT), followed by incubation with a secondary HRP-conjugated antibody and Opal Fluorophore Working Solution (TSA, 1 : 100) for 10 min at RT. For Opal IHC, primary antibodies included antibodies against HIF-1α (1 : 100; Proteintech, USA), PIK3CB (1 : 100; Abcam, UK), and F2RL1 (1 : 100; Abcam, UK). Tissue slides were imaged using the Mantra Quantitative Pathology Imaging Systems (PerkinElmer, USA). Image analysis was performed with the InForm image analysis software (PerkinElmer, USA).