Fig. 4: Decreased carbon flux through PDH is Hif1α independent.
a Immunoblot (IB) for HIF1α protein of three independent nuclear extracts from WT and Nos2−/− BMDMs stimulated for 24 h. Lamin B was used as loading control (n = 6). b IB for PDH-Phospho-Ser293 in mitochondrial extracts; citrate synthase (CS) was used as loading control (n = 6). c O.N.-treated WT and Hif1α−/− BMDMs were seeded in Seahorse XF96 cell culture plates and sequential treated with oligomycin (Oligo), FCCP, and rotenone plus antimycin A (Rot/AA). Basal OCR and extracellular acidification rate (ECAR) are quantified (n = 3). Data were not significant by two-way ANOVA with Sidak’s post-tests (p-values = 0.59, 0.1675). d Heat-maps from GC-MS analysis of metabolites of glycolysis and TCA cycle from Hif1α−/− BMDMs. Data show log10 ratio from the average peak areas of stimulated cells compared to unstimulated. Data (n = 4) were analyzed by two-way ANOVA (interaction < 0.0001) (Sidak’s post-tests). e Representative Seahorse analysis of pyruvate/malate respiration in WT and Hif1α−/− BMDMs. Bar graphs show quantified OCR (n = 3) (two-way ANOVA, interaction p = 0.036, Sidak’s post-tests). f Protein normalized basal OCR from stress tests of WT and Nos2−/− macrophages treated with Dimethyl α-KG (D-αkG) 1 h prior to O.N. stimulation. Data were analyzed by Student’s t-test (n = 3). g BMDMs as in f and stimulated for 8 h. mRNA from total cell lysates was analyzed by qPCR for Pdk1. Data (n = 6) were not significant by two-way ANOVA (interaction p = 0.74) (Sidak’s post-tests). h Basal OCR/ECAR in WT and Nos2−/− BMDMs stimulated O.N. and pretreated with DMOG. Data (n = 3) were analyzed by two-way ANOVA (interaction < 0.0001) (Sidak’s post-tests). i Enzymatic activity of PDH in total cell lysates from WT and Nos2−/− BMDMs after O.N. activation. Data (n = 6) were analyzed by unpaired t-test with Welch’s correction (p = 0.0038). j PDH-E3 (DLD) in-gel activity assay and IB on native gels of mitochondrial fractions from macrophages from two independent WT and Nos2−/− mice. k Mitochondrial fractions from control and LPS + IFNγ stimulated WT and Nos2−/− BMDMs were used as inputs to immunoprecipitate DLD. Anti DLD and Anti-CysSNO IBs were performed. All error bars display mean ± SEM. Source data are provided as a Source Data file.
