Fig 2: miR-17~92 directly targets PHD2. A, The putative binding site of miR-17 and miR-20a in the 3'-UTR of PHD2 and PHD3. B, The expression levels of PHD1, PHD2, and PHD3 proteins were determined in cell lysates of normal hPASMC infected with control lentiviruses (pLVX/Ctrl) or lentiviruses encoding the whole cluster of miR-17~92 (miR-17~92). Tubulin was used as the internal control. C, We determined the protein levels of PHD2 in freshly isolated mPASMC from miR-17~92fl/fl and sm-17~92-/- mice. In each strain mPASMC from at least 3 mice were subjected to Western blotting. Representative blots are shown in the top panel and quantification in the bottom panel. D through F, Normal hPASMC were treated with miR-17 and miR-20a mimics as well as negative control (Neg) and harvested 48 hours after transfection to determine the levels of PHD1 (D), PHD2 (E), and PHD3 (F). The amount of tubulin was used as loading control. n=3. G through I, Normal hPASMC were treated with miR-17 and miR-20a inhibitors or negative control (Neg) and harvested 48 hours after transfection to determine the levels of PHD1 (G), PHD2 (H), and PHD3 (I). The amount of tubulin was used as loading control. n=5. J, Diagram of the wild-type (Wt-Luc) and mutated (Mut-Luc) 3'-UTR of PHD2 luciferase reporter constructs. Open triangle represents the binding site of miR-17/20a, and the solid triangle represents the mutated miR-17/20a binding site. K through N, Normal hPASMC were cotransfected with the Wt-Luc reporter (K and L) or Mut-Luc (M and N) and inhibitors (K and M) or mimics (L and N) of miR-17 and miR-20a. n=3 for C through I, 9 for K and L, 5 for M, 4 for N. hPASMC indicates human pulmonary artery smooth muscle cell; mPASMC, mouse pulmonary artery smooth muscle cell; PHD, prolyl hydroxylase. *P<0.05; **P<0.01. Data are presented as mean±SEM.

Fig 3: Non-hypoxic stabilization of hypoxia-inducible factor 1-alpha (HIF-1a) induces glycolysis and rescues ETC inhibitor-induced reduction in cytokine production and cell death in tissue-resident alveolar macrophages (TR-AMs).TR-AMs were treated (16 hr) overnight ±FG-4592 (25.0 µM when not stated otherwise). (A) Glycolysis was measured as extracellular acidification rate (ECAR). (B) Quantification of glycolytic parameters. Data represent at least three independent experiments (n = 4 separate wells per group). Glycolytic parameters compared to control group (0.0 µM) and significance was determined by one-way ANOVA with Bonferroni correction. (C) Western blot analysis of nuclear extract for HIF1a expression and (D) whole cell lysate for glycolytic enzyme and prolyl hydroxylase expression. (E) Mitochondrial stress test to measure oxygen consumption rate (OCR). (F) Quantification of mitochondrial respiration parameters. Data represents at least three experiments (n = 4 separate wells per group). Mitochondrial parameters were compared to control group (0.0 µM) and significance was determined by one-way ANOVA with Bonferroni correction. (G) ECAR measurement during mitochondrial stress test. (H) TR-AMs were pretreated overnight (16 hr) with 0.0 µM (no treatment) or 25.0 µM FG-4592, then stimulated with 20 ng/ml lipopolysaccharide (LPS) in the presence or absence of mitochondrial inhibitors (20 nM antimycin A [Ant] or rotenone [Rot]) for 6 hr while maintaining pretreatment conditions. Sandwich ELISA was used to measure secreted cytokine (TNFa, IL-6, KC, and CCL-2). Data represents at least three independent experiments; n = 3 per group. (I) TR-AMs were treated with FG-4592 for 6 hr, then treated with mitochondrial inhibitors (100 nM Ant or 500 nM Rot) overnight and a sulforhodamine B assay was performed to measure cytotoxicity. Bar graphs represent cytotoxicity compared to control, 0.0 µM group. Data represents at least three independent experiments (n = 3 per group). Significance was determined by two-way ANOVA with Bonferroni correction. All error bars denote mean ± SD. *p<0.05. Figure 6—source data 1.The effect of FG-4592 on hypoxia-inducible factor 1-alpha (HIF-1a) expression in tissue-resident alveolar macrophages (TR-AMs).Uncropped Western blot images of HIF-1a in TR-AMs treated with FG-4592 or control vehicle. Figure 6—source data 2.The effect of FG-4592 on glycolytic enzyme and prolyl hydroxylase protein expression in tissue-resident alveolar macrophages (TR-AMs).Uncropped Western blot images of HK2, LDHA, PHD2, PHD3, and a-tubulin in TR-AMs treated with FG-4592 or control vehicle.

Fig 4: Phd3 interference promotes HIF-1, fibrosis and inflammation in WT AT. 12-week-old female C57/Bl6 mice (n = 7) were injected with Phd3 interference adenovirus (shPHD3) in one inguinal fat pad and a control vector (shNS) in the contra-lateral pad. qPCR quantification of (A) Phd3 and Era, (B) Lox, Col1a1 and Col3a1 Col6a1 and (C) TNFa, IL6 and IL1ß and TLR4 mRNA in inguinal ATs. Data shown as mean ± s.e.m. *P < 0.05 by two-tailed Student's t-test relative to shNS. See also Supplementary Figures 3 and 4.

Fig 5: PHD2/3 bind to CPT1B and regulate LCFA uptake and oxidation in primary cardiomyocytes(A) Coomassie-stained SDS-PAGE of FLAG-PHD3 co-immunoprecipitated proteins in NRVMs. PHD3 or LacZ was overexpressed in NRVMs, and co-immunoprecipitation was performed with anti-FLAG agarose beads. Pulled-down proteins were separated with SDS-PAGE and visualized with G-250 Coomassie blue. Protein identities were determined by tandem mass spectrometry analysis (1. Dync1h1; 2. Na+/K+ ATPase a-1; 3. ATP synthase B; 4. GAPDH; 5. ANT; 6. MLP). The respective positions of PHD3 and CPT1B are labeled in the figure.(B) Co-immunoprecipitation and western blot revealed PHD2 and 3 binding to CPT1B. FLAG-PHD2 and FLAG-PHD3 were immunoprecipitated by anti-FLAG beads, and the precipitated proteins were analyzed by western blot with anti-FLAG and anti-CPT1B antibodies.(C) Co-immunoprecipitation and western blot revealed endogenous CPT1B binds to endogenous PHD2 and 3. The precipitated proteins were analyzed by western blot with anti-PHD2, PHD3, or CPT1B antibodies.(D) Oxygen consumption rate (OCR) in primary cardiomyocytes after DMOG or Etomoxir (CPT1 inhibitor, Eto) treatment. OCR was real-time analyzed in living NRVMs by using a SeaHorse XFp Flux Analyzer. DMOG treatment leads to a drastic decrease in LC-FAO.(E) Maximal respiration rate was estimated for each experimental group of cells.(F and G) Similar experiment was conducted by using PHD2/3_HKO NMCMs. After 4 consecutive days of 4-hydroxyl-tamoxifen (4OHT) treatment to induce PHD2/3 disruption (or DMSO for untreated control cells), OCR was measured following the same protocol that described for NRVMs (F) and maximal respiration rate estimated afterward (G). A dramatic decrease in LC-FAO was found in PHD2/3-deficient cardiomyocytes, and maximal respiration rate was significantly decreased only in 4OHT treated PHD2/3_HKO NMCMs therefore in response to the loss of PHD2/3.(H) Mitochondrial LCFA uptake in primary cardiomyocytes after DMOG treatment. Five days after seeding, NRVMs were cultured in serum free medium with 1 µM BODIPY 500/510 C12 in the presence of DMOG (1mM) or DMSO for 16 h (H, left panel). Mitochondria were labeled 5 with Mitotracker Red (H, middle panel).(I) The same overlay of each experiment at a higher magnification.Scale bars, 50 µm. For (D)–(G), results are shown ±SEM for an average of at least 4 different experiments: ****p < 0.0001.