(A) GST pull-down assays were performed with GST or GST-HIF-1α (531–826) and lysate prepared from HEK293 cells grown in media containing light isotopes (¹²C₆-¹⁴N₂-Lys and ¹²C₆-¹⁴N₄-Arg) or heavy isotopes (¹³C₆-¹⁵N₂-Lys and ¹³C₆-¹⁵N₄-Arg), respectively. The precipitated proteins were analyzed by Q-TOF mass spectrometry (Luo et al., 2010). The mass spectrum of a tryptic PKM2 peptide is shown. Monoisotopic peaks derived from PKM2 bound to GST-HIF-1α (531–826) are 4 Da greater in their mass/charge ratio (m/z) and have higher relative intensity compared to the corresponding peaks due to non-specific binding of PKM2 to GST.
(B) Co-IP was performed in HeLa cells exposed to 1% O₂ for 24 h.
(C) Co-IP was performed using nuclear lysates (NL) prepared from HeLa cells transfected with PKM2-V5 vector and exposed to 1% O₂ for 24 h.
(D and E) GST pull-down assays were performed with GST or GST fusion protein containing the indicated amino acid residues of HIF-1α (upper panels) and whole cell lysates (WCLs) from HeLa cells expressing PKM2-V5. bHLH, basic helix-loop-helix; PAS, Per-ARNT-Sim; TAD, transactivation domain; ID, inhibitory domain.

(A) GST-E10 was incubated with WCLs to induce hydroxylation and tryptic peptides were analyzed by mass spectrometry. The fragmentation spectrum of ⁴⁰¹LAPITSDPTEATAVGAVEASFK⁴²² revealed the presence of peptides with hydroxylation of Pro-403 or Pro-408.
(B) HEK293 cells were transfected with WT FLAG-HIF-1α or double mutant (DM) FLAG-HIF-1α(P402A/P564A) and treated with the proteasome inhibitor MG132 (10 μM) for 4 h. IP was performed with anti-FLAG antibody, followed by immunoblot assay. The intensity of hydroxylated FLAG-HIF-1α was quantified by densitometry and normalized to WT.
(C) HeLa cells were transfected with vector encoding PKM2-V5 or PKM1-V5 and exposed to 20% or 1% O₂ for 24 h. IP was performed with anti-V5 agarose, followed by immunoblot assay.
(D) HeLa cells were exposed to 1% or <0.1% O₂ for 4 h. IP was performed with anti-PKM2 antibody, followed by immunoblot assay. The intensity of hydroxylated PKM2 was quantified by densitometry and normalized to 1% O₂.
(E) HeLa cells were transfected with vector encoding WT PKM2-V5 or PKM2(P403/408A)-V5 and exposed to 20% or 1% O₂ for 24 h. IP was performed with anti-V5 agarose, followed by immunoblot assay.
(F and G) HeLa cells were co-transfected with p2.1 (F) or GalA(P564A) and pG5E1bLuc (G); pSV-Renilla; and the indicated expression vector, and exposed to 20% or 1% O₂ for 24 h. The ratio of FLuc:RLuc activity was normalized to EV at 20% O₂ (mean ± SEM, n = 4). ^***p<0.001 vs EV; ^#p<0.05, ^###p<0.001 vs PKM2(WT).
(H) Co-IP was performed in HeLa cells transfected with vector encoding WT PKM2-V5 or PKM2(P403/408A)-V5 and exposed to 1% O₂ for 4 h.
(I) HeLa cells were transfected with PKM2-V5 vector and treated with DMSO or DMOG (100 μM) for 4 h. IP was performed with anti-V5 agarose, followed by immunoblot assay.
(J) Co-IP was performed in HeLa cells co-transfected with FLAG-HIF-1α(DM) and PKM2-V5 vectors and treated with DMSO or DMOG for 4 h.
(K) HeLa cells were co-transfected with GalA(N803A) vector, pG5E1bLuc, pSV-Renilla, and EV or PKM2-V5 vector, and treated with DMSO or DMOG for 24 h. The ratio of FLuc:RLuc activity was normalized to EV + DMSO (mean ± SEM, n = 4). ^*p<0.05 vs EV; ^#p<0.05, ^##p<0.01 vs DMSO.
See also Figure S3.

(A) HeLa cells were transfected with shSC or shPKM2-#1 vector and exposed to 20% or 1% O₂ for 4 h. ChIP assays were performed with IgG, anti-HIF-1α, or anti-HIF-2α antibody (mean ± SEM, n = 4). ^#p<0.05 vs shSC-20% O₂; ^*p<0.05 vs shSC-1% O₂.
(B) HeLa cells were transfected with shSC or shPKM2-#1 vector, and exposed to 1% O₂ for 4 h. ChIP assays were performed with IgG or anti-HIF-1β antibody (mean ± SEM, n = 3). ^#p<0.05 vs IgG; ^*p<0.05 vs shSC.
(C and D) HeLa cells were exposed to 1% O₂ for 24 h. ChIP assays were performed with IgG, anti-PKM2 (C), or anti-PHD3 (D) antibody (mean ± SEM, n = 3). ^**p<0.01, ^***p<0.001 vs IgG.
(E) Co-IP was performed in HeLa cells transfected with PKM2-V5 vector for 24 h.
(F) HeLa cells expressing shSC or shPKM2-#1 were exposed to 20% or 1% O₂ for 24 h. ChIP assays were performed with IgG or anti-p300 antibody (mean ± SEM, n = 3). ^*p<0.05, ^**p<0.01 vs shSC; ^###p<0.001 vs shSC-20% O₂.
(G) HeLa cells were transfected with shSC or shPKM2-#1 vector and exposed to 1% O₂ for 24 h. ChIP assays were performed with IgG or anti-acetylated H3K9 (H3K9-ac) antibody (mean ± SEM, n = 3). ^**p<0.01 vs shSC.
(H) Immunoblot assays of total H3K9-ac, histone H3, and PKM2 in HeLa cells transfected with shSC or shPKM2-#1 vector and exposed to 1% O₂ for 24 h.
See also Figure S5.

(A) qRT-PCR analysis of Pkm1, Pkm2 and Slc2a1 mRNA in wild-type (WT) and HIF-1α knockout (KO) mouse embryo fibroblasts (MEFs) exposed to 20% or 1% O₂ for 24 h (mean ± SEM, n = 3–5). ^***p<0.001 vs 20% O₂. ^###p<0.001 vs WT MEFs.
(B) Immunoblot assays of HIF-1α, PKM2, histone H3, and α-tubulin protein in nuclear and cytosolic lysates prepared from HeLa cells exposed to 20% or 1% O₂ for 24 h. The intensity of PKM2 was quantified by densitometry and normalized to 20% O₂ (mean ± SEM, n = 3–4). ^*p<0.05, ^**p<0.01 vs 20% O₂.
(C) Nucleotide sequence of the hypoxia-response element (HIF-1 binding site 5′-ACGTG-3′ and 5′-CACA-3′ site shown in red) within intron 1 of the human PKM2 gene. The transcription initiation site is designated +1. Exons and intron are not drawn to scale.
(D) Chromatin was precipitated from DMOG-treated HeLa cells (4 h) with antibodies against HIF-1α, HIF-2α, or IgG, and analyzed by qPCR (mean ± SEM, n = 4–5). *p<0.05 vs IgG.
(E and F) HeLa cells were co-transfected with pGL2p control (CON), pGL2p-WT PKM2 HRE (wtHRE), or pGL2p-mutant PKM2 HRE (mutHRE) with CGT→AAA (E) or CAC→AAA (F), and pSV-Renilla, and exposed to 20% or 1% O₂ for 24 h. The ratio of FLuc:RLuc activity was normalized to CON at 20% O₂ (mean ± SEM, n = 4). ^#p<0.05, ^###p<0.001 vs CON; ^***p<0.001 vs wtHRE.
(G) HeLa cells were transfected with: vector encoding a short hairpin (sh) RNA targeting HIF-1α, HIF-2α, or a scrambled control (SC); pGL2p-wtHRE; and pSV-Renilla, and exposed to 20% or 1% O₂ for 24 h. The ratio of FLuc:RLuc activity was normalized to shSC at 20% O₂ (mean ± SEM, n = 4). ^***p<0.001 vs shSC.
See also Figure S1.

(A, B, G and I) HeLa cells were transfected with p2.1, pSV-Renilla, and empty vector (EV) or the indicated expression vector, and exposed to 20% or 1% O₂ for 24 h. The ratio of FLuc:RLuc activity was normalized to EV (A, G and I) or shSC (B) at 20% O₂ (mean ± SEM, n = 4). ^*p<0.05, ^**p<0.01, ^***p<0.001 vs EV or shSC.
(C) Immunoblot assays of HIF-1α, HIF-1β, PKM2-V5, and actin in HeLa cells transfected with EV or PKM2-V5 (M2) expression vector and exposed to 20% or 1% O₂ for 4 h.
(D) HeLa cells were transduced with a retrovirus encoding shSC or two retroviruses encoding different shRNAs targeting PKM2 (shM2), exposed to 20% or 1% O₂ for 4 h, and WCLs were subjected to immunoblot assays.
(E, F and H) HeLa cells were transfected with GalA or GalO, pG5E1bLuc, and pSV-Renilla, and the indicated expression vector, and exposed to 20% or 1% O₂ for 24 h. The ratio of FLuc:RLuc activity was normalized to GalO (E and F) or EV (H) at 20% O₂ (mean ± SEM, n = 4). ^*p<0.05, ^**p<0.01, ^***p<0.001 vs EV or shSC.
(J) GST pull-down assays were performed with GST, GST-E9, or GST-E10 and WCLs from HeLa cells expressing FLAG-HIF-1α (531–826).
See also Figure S2.

(A) Co-IP was performed in HeLa cells co-transfected with PKM2-V5 and PHD3 vectors.
(B) GST pull-down assays were performed with purified GST, GST-E9, or GST-E10 and WCLs from HeLa cells exposed to 1% O₂ for 24 h.
(C) HeLa-shSC, HeLa-shPHD3-704, and HeLa-shPHD2 cells were transfected with PKM2-V5 vector and exposed to 20% or 1% O₂ for 24 h. IP was performed with anti-V5 agarose, followed by immunoblot assay. Hydroxylated PKM2-V5 was quantified by densitometry and normalized to total immunoprecipitated PKM2-V5 (mean ± SEM, n = 3). ^**p<0.01, ^***p<0.001 vs shSC.
(D) RCC4 cells were co-transfected with GalA(P564A) vector, pG5E1bLuc, pSV-Renilla, and the indicated expression vector for 24 h. The ratio of FLuc:RLuc activity was normalized to EV (mean ± SEM, n = 4). ^*p<0.05; ^**p<0.01; ^***p<0.001. mutPHD3, PHD3(H135A/D137A); mutPKM2, PKM2(P403/408A).
(E) HeLa cells were co-transfected with GalA(P564A) vector, pG5E1bLuc, and pSV-Renilla, and the indicated expression vector and exposed to 20% or 1% O₂ for 24 h. The ratio of FLuc:RLuc activity was normalized to shSC at 20% O₂ (mean ± SEM, n = 4). ^***p<0.001 vs shSC.
(F) Co-IP was performed in HeLa cells co-transfected with vector encoding double mutant (DM) FLAG-HIF-1α(P402A/P564A), EV or PHD3, and PKM2-V5.
(G) Co-IP was performed in HeLa cells co-transfected with vectors encoding PKM2-V5 and either shSC or shPHD3 and exposed to 1% O₂ for 24 h.
See also Figure S4.

(A) qRT-PCR analysis of indicated mRNAs in HeLa cells transfected with shSC or shPKM2-#1 vector and exposed to 20% or 1% O₂ for 24 h (mean ± SEM, n = 4). ^*p<0.05, ^**p<0.01, ^***p<0.001.
(B) HeLa cells were transduced with shSC or shPKM2-#1 and shPKM2-#2 retrovirus, and exposed to 20% or 1% O₂ in the absence or presence of digoxin (100 nM) for 24 h. Levels of the indicated protein were determined by immunoblot assay, quantified by densitometry, and normalized to actin (mean ± SEM, n = 4). ^*p<0.05, ^***p<0.001.
(C) qRT-PCR analysis of indicated mRNAs in HeLa cells transduced with shSC or shPHD3-704 retrovirus and exposed to 20% or 1% O₂ for 24 h (mean ± SEM, n = 3–4). ^###p<0.001 vs shSC-20% O₂; ^**p<0.01, ^***p<0.001 vs shSC-1% O₂.
(D–F) RCC4 cells were transduced with shSC or shPHD3-704 retrovirus. Glucose was measured in lysates and normalized to total cellular protein amount (mean ± SEM, n = 3; D); lactate was measured in the culture media and normalized to cell number (mean ± SEM, n = 3; E); O₂ consumption rate (OCR) was measured and normalized to cell number (mean ± SEM, n = 4–8; F). ^*p<0.05, ^**p<0.01, ^***p<0.01 vs shSC.
(G) PKM2 is prolyl hydroxylated by PHD3 and interacts with HIF-1α and p300, thereby enhancing HRE occupancy by HIF-1 and p300, and increasing histone acetylation.
(H) Feed-forward mechanism for HIF-1 activity. HIF-1 activates transcription of genes encoding PHD3 and PKM2, which interact with HIF-1α to stimulate transactivation of target genes encoding GLUT1, LDHA, PDK1, and other metabolic enzymes that mediate the Warburg effect in cancer cells.
See also Figure S6.