Ponceau s staining

Aliquots saved from the experiment were quantified using the Bio-Rad protein assay reagent, and an equal quantity of protein was loaded onto 10% SDS-polyacrylamide gel electrophoresis gel and separated by electrophoresis. Proteins were transferred to a poly(vinylidene difluoride membrane (Bio-Rad) and confirmed with Ponceau S staining (Sigma) following manufacturer’s instructions. Blots were blocked with 5% dry milk in TBST (0.1% Tween-20 in Tris-buffered saline) overnight at 4 °C, washed extensively with TBST, then incubated with 1:5000 streptavidin-HRP (Thermo Fisher Scientific) in 5% BSA in TBST for 1 h at room temperature. Immunoblots were washed again with TBST then detected using enhanced chemiluminescence reagents (Thermo Fisher Scientific) and imaged using a Syngene G:BOX Chemi system.

A total of 50 μg of protein was extracted from normoxic, hypoxic, and PHDI-treated CDCs using lysis buffer containing protease inhibitors (Sigma-Aldrich), and subjected to immunoblot assay with anti-HIF-1α (1:1,000; Novus), anti-citrate synthase (1:1,000; Alpha Diagnostic) and anti-GLUT-1 (1:1,000; Abcam), as previously described (24 (link)). Protein loading and transfer were confirmed by Ponceau S staining (Sigma-Aldrich), and protein levels were normalized to internal standard (rat heart lysates) to ensure homogeneity between samples and gels. Bands were quantified using UN-SCAN-IT gel software (Silk Scientific), and all samples were run in duplicate on separate gels to confirm results.

Cells were lysed with RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP40) in the presence of protease and phosphatase inhibitors. For subcellular fractionation, 2 × 10⁸ cells were collected for the extraction and both NE-PER nuclear and cytoplasmic extraction kits (Thermo-Scientific, Waltham, MA) were used. Prior to SDS-PAGE, cell lysates were re-suspended in SDS sample buffer (60 mM Tris–HCl, 1% SDS, 10% glycerol, 0.05% bromophenol blue, pH 6.8, with 2% β-mercaptoethanol). Samples were subjected to 10% SDS-PAGE (Bio-Rad, Hercules, CA) and transferred to PVDF membranes (Bio-Rad). Transfer efficiency was determined by Ponceau S staining (Sigma-Aldrich). PVDF membranes were incubated with blocking solution (TBS containing 0.1% Tween 20 and 5% BSA) and were probed with specific antibodies (anti-PanH4ac (rabbit): # 39925 Active motif, Carlsbad, CA; anti-pan H4ac (mouse): 61337 #anti-PanH3ac: # 61638 Active motif; anti-H3 total: #61648 Active motif; anti-H4K12ac: # 39165 Active motif; anti-K5ac: # 39699 Active motif; anti-K8ac: # 2594S Cell Signaling, Danvers MA; anti-K16ac # 13534S Cell signaling; anti-GAPDH: Santa Cruz Biotechnology, Dallas, TX; anti-BRD4: Abcam Cambridge UK). Protein bands were detected using chemiluminescence kit (Millipore, Billerica, MA). ImageJ was used to quantify immunoblot results.