Figure 2.

Pyruvate kinase isoform M2 (PKM2) and hypoxia-inducible factor 1α (Hif-1α) bind directly to the promoter region of PDL1 in primary murine BMDM cells. BMDM cells were treated with TEPP-46 at 50 µM for 1 h prior to stimulation with LPS (100 ng/ml, 24 h) (A). Binding of PKM2 (left) or Hif-1α (right) to HRE1 or HRE4 of the PD-L1 promoter was detected by incubating cell lysates with biotinylated oligonucleotides spanning the relevant HRE promoter region. Protein–oligonucleotide complexes were isolated using streptavidin agarose beads, and proteins were detected by western blotting. Representative of n = 3. Chromatin immunoprecipitation (ChIP)-PCR (B) using PKM2 and HIF-1α antibodies and primers specific for three promoter regions of PD-L1 (HRE1, 2–3, and 4) and a known Hif-1α binding region of vascular endothelial growth factor as a positive control showing binding of Hif-1α (left) and PKM2 (right) to the PD-L1 promoter in LPS-treated BMDMs (100 ng/ml, 24 h). (C) Sequential ChIP assays measuring simultaneous endogenous binding of PKM2 and Hif-1α to chromatin in response to LPS (100 ng/ml, 24 h) ±TEPP-46 (pretreatment using 50 µM, 60 min). ChIP data are calculated as percent of input, error bar represents mean ± SEM, and statistics are performed as two-tailed unpaired t-test *P < 0.05, **P < 0.01, and ***P < 0.001.