Gtx127309

Cellular proteins were extracted using RIPA lysis buffer (G2002, Servicebio, Wuhan, China), and protein concentrations were determined using a BCA kit (G2026, Servicebio, Wuhan, China). Subsequently, proteins were separated through electrophoresis and transferred onto polyvinylidene difluoride (PVDF) membranes (IPVH00010, Millipore, Burlington, Massachusetts, USA). After blocking with 5% non-fat milk, the membranes were incubated with specific primary antibodies at 4 °C overnight, followed by incubation with secondary antibodies for 1 h at 25 °C. Chemiluminescence reagent (G2014, Servicebio, Wuhan, China) was employed for signal detection using the Clinx imaging system (Shanghai, China). Primary antibodies against P4HA1 (1:1000, 12658-1-AP) and β-actin (ACTB, 1:1000, 20536-1-AP) were purchased from Proteintech (Wuhan, China), and against TET2 (1:1000, A5682), FBP1 (1:1000, A11664), P4HA2 (1:1000, A22150), P4HA3 (1:1000, A13767), P4HB (1:1000, A19239) from AbClonal (Wuhan, China). The primary antibody for hypoxia-inducible factor-1α (HIF-1α, GTX127309) was purchased from Gene Tex (Irvine, CA, USA). The primary antibody for Flag (1:1000, MA1-91878) was purchased from Thermo Scientific (USA). Gastrocnemius muscle tissue proteins were extracted using the same procedure followed by homogenization.

For murine skin IF samples, immediately after sacrifice, mouse skin tissues were fixed with periodate-lysine-paraformaldehyde fixative overnight. Samples were then dehydrated with 30% sucrose, embedded in OCT compound (Tissue-Tek), and stored at −80°C. Sections were cut at 7 μm and stained with the following Abs: anti–mouse CD4 (clone RM4-5; BioLegend, catalog 100547; 1:100), anti–mouse CD8a (clone 53-6.7; BioLegend, catalog 100747; 1:100), anti-pimonidazole mouse FITC- or DyLight 549-Mab (clone 4.3.11.3; Hypoxyprobe, catalog HP6-200 or HP7-200; 1:100), rabbit polyclonal anti–HIF-1α (GeneTex, catalog GTX127309; 5 μg/mL) or rabbit polyclonal isotype control (GeneTex, catalog GTX35035; 5 μg/mL), and Alexa Fluor 647–conjugated goat anti-rabbit secondary Ab (Thermo Fisher Scientific, catalog A-21244; 1:1,000). Confocal microscopy was conducted with a Leica SP8 (Figure 1 and Supplemental Figure 2) or SP5 (Supplemental Figure 3) laser-scanning confocal microscope at the Cell Imaging Core, Yale Stem Cell Center.

In IHC, paraffin blocks of the xenograft tumors were cut into 4-μm sections. The slides were incubated with primary antibodies: IGFBP3 (1:200, MAB305; R&D Systems, Minneapolis, MN, USA), HIF-1α (1:500, H1alpha67-ChIP Grade; Abcam, Cambridge, UK), HIF-2α (1:1,000, ab8365; Abcam, Cambridge, UK), HO-1 (1:200, GTX101147, Genetex, Irvine, CA, USA), or Von Hippel-Lindau (VHL) (1:200, GTX101087, Genetex, Irvine, CA, USA) at 4 °C overnight (about 17 h).
In ICC, 0.2 × 10⁵ cells were cultured on 25 mm × 25 mm glass slides and incubated under hypoxic or normoxic conditions for 17 h. Then, the slides were incubated with primary antibodies: IGFBP3 (1:200; MAB305; R&D Systems, Minneapolis, MN, USA), HIF-1α (1:300; GTX127309; Genetex, Irvine, CA, USA), or HIF-2α (1:300; GTX30114; Genetex, Irvine, CA, USA) at 4 °C overnight (about 17 h).
A chromogen in IHC and ICC was developed, following the protocol of UltraVision Quanto Detection System HRP DAB (Thermo Fisher Scientific, Waltham, MA, USA). The sections or slides were stained with hematoxylin for examination, and photos were taken using Moticam X3 Plus (Motic, Speed Fair Co., Ltd, HK) microscope camera by Motic Images Plus 3.0 software. The signals of IHC and ICC were quantified and analyzed by ImageJ 1.53 k software (NIH, USA).

The antibodies used in this study are as follows: anti-active-caspase-3 rabbit polyclonal antibody (G7481, Promega), anti-Bax mouse monoclonal antibody (6A7, Sigma-Aldrich), anti-hBid rabbit polyclonal antibody (AF846, R&D, Minneapolis, MN), anti-Bak mouse monoclonal antibody (MAB8161, R&D), anti-HA tag antibody (HA.11/16B12, Covance Inc., Princeton, NJ), anti-Green fluorescent protein monoclonal antibody (mFX73, Wako, Osaka, JAPAN), anti-HIF1 alpha Rabbit polyclonal antibody (GTX127309, GeneTex, Irvine, CA) and anti-cytochrome-c Rabbit polyclonal antibody (GTX108585, GeneTex).

Extracted protein (1 mg) was immunoprecipitated overnight with HIF-1α (GTX127309; GeneTex) or YAP antibody (#ab52771; Abcam). The following day, the mixture was incubated with protein A/G magnetic beads (LSKMAGAG02; Millipore, Burlington, MA, USA) at 4 °C for 1 h. The supernatant was discarded, and the beads were washed twice with cold PBS containing 0.1% Tween 20 and boiled in sample buffer (10% SDS, 1 M Tris-HCl, 30% glycerol, 6% 2-mercaptoethanol, and 0.12% bromophenol blue in RIPA buffer) for 10 min. The boiled supernatant was transferred to a clean eppendorf tube and subjected to Western blot analysis. The protocol for the negative IgG control groups was the same as the above, except that the antibody used for immunoprecipitated overnight was IgG antibody (#ab172730; Abcam).