Fig 2: Hif2a-dependent induction of amphiregulin during myocardial injury. a, b Myosin-Cre+ or Hif2aloxP/loxPMyosin-Cre+ underwent 45 min myocardial ischemia, followed by 120 min reperfusion. After harvest of the area-at-risk, total RNA was probed for transcriptional changes using a microarray technique (Agilent-014868 Whole Mouse Genome Microarray 4x44K G4122F Probe Name version). Transcriptional changes were calculated in relation to the myocardial baseline expression in untreated littermate controls of the same genotype. Array data have been deposited at http://www.ncbi.nlm.nih.gov/geo/ (accession number GSE67308). a List of “top hits” of genes transcriptionally induced in the Area-At-Risk in Myosin-Cre+ but unchanged in Hif2aloxP/loxPMyosin-Cre+ (n = 4 per group, mean ± SD). Note: the most prominent differential regulation was found for the epidermal growth factor amphiregulin (Areg). b Analysis of relevant networks in the IPA (Ingenuity Pathway Analysis®) library most significant to the dataset. Note: selective induction of Areg c Myosin-Cre+, Hif1aloxP/loxPMyosin-Cre+, or Hif2aloxP/loxPMyosin-Cre+ underwent 60 min myocardial ischemia, following 120 min reperfusion. Total RNA from the area-at-risk was probed for transcriptional changes of Areg by real-time RT-PCR. Transcriptional changes were calculated relative to an internal housekeeping gene (Actb). Data are expressed as mean fold change ± SD compared to untreated myocardial tissue from littermates of the respective genotype compared by one-way ANOVA followed by Bonferroni’s multiple comparison test (Myosin-Cre+: -I n = 6, +I n = 5; Hif1aloxP/loxPMyosin-Cre+: -I n = 3; +I n = 4; Hif2aloxP/loxPMyosin-Cre+: -I n = 6; +I n = 7; F5,25 = 33.53; p < 0.0001). d AREG immunoblot performed for protein isolated from the ischemic myocardial tissue after 60 min of ischemia and 120 min reperfusion or untreated littermate controls of Myosin-Cre+, Hif1aloxP/loxPMyosin-Cre+ or Hif2aloxP/loxPMyosin-Cre+, respectively. A representative image of three individual experiments is presented. e Quantification by densitometry of the AREG immunoblot results relative to ACTb. Data are expressed as mean fold change ± SD normalized to untreated myocardial tissue from Myosin-Cre+ compared by one-way ANOVA followed by Bonferroni’s multiple comparison test (n = 3 per group; F5,12 = 4.487; p = 0.0154)

Fig 3: HIF is essential to tumor growth by VHL-deficient RCC cells.A. Total cell lysates of human renal carcinoma 786-O cells stably expressing the control shRNA (SCR) or the HIF1b-1770 were prepared and immunoblotted with the indicated antibodies. B. 786-O VHL-/- cells infected to produce SCR or HIF1b-1770 were injected subcutaneously into the flanks of nude mice. Approximately 8-10 weeks later, tumors were excised and weighed. Thirteen tumors per line were analyzed. Error bars = standard error of the mean. P<=0.001 according to a Mann-Whitney U statistic analysis. C. Representative photographs of tumors analyzed in Figure 1B. Left: Tumor from cells expressing SCR; Right: Tumor from cells expressing HIF1b-1770. D. Total cell lysates of 786-O cells stably expressing the SCR or the HIF2a-566 were immunoblotted with the indicated antibodies. E. 786-O VHL-/- cells infected to produce SCR or HIF2a-566 were injected subcutaneously into the flanks of nude mice. Approximately 8-10 weeks later, tumors were excised and weighed. Ten tumors per line were analyzed. Error bars = standard error of the mean. P<=0.001 according to a Mann-Whitney U statistic analysis. F. Representative photographs of tumors analyzed in Figure 1E. Left: Tumor from cells expressing SCR; Right: Tumor from cells expressing HIF2a-566.

Fig 4: 4-OOHCPA exposure induced HIF2A.A: Western blot analysis of HIF2A protein (118KD) and actin (43KD) in limbs at 10 min, 1, 3, 6, and 24 h after treatment with 4-OOHCPA at 0.3 µg/ml (L) 1.0 µg/ml (M) or 3.0 µg/ml (H). P represents the positive control. B: Fold changes from the scan densitometry quantification of the HIF2A band in drug treated groups compared to control groups. Hatched bar: 0.3 µg/ml (L), cross-hatched bar: 1.0 µg/ml (M) and gray bar: 3.0 µg/ml (H). Each bar (mean ± SEM) represents six to seven replicates; bars with an asterisk are significantly different from control at the same time point (p =0.05).

Fig 5: Divergent gene expression in dopamine-depleted mice is largely restricted to GPe PV+ neurons and implicates the HIF pathway. A, Numbers of genes that have differential expression with DD in each cell type. DD was confirmed by immunochemistry (Extended Data Figure 4-1), and RNA-seq sample quality was confirmed computationally (Extended Data Figure 4-2). B, Volcano plot of DD-affected genes in GPe PV+ neurons. Orange represents genes meeting the significance threshold of padj < 0.05, listed in order of significance. See Extended Data Figure 4-3 for a detailed list of differentially expressed genes and Extended Data Figure 4-5 for functional relationships. C, Dopamine depletion is associated with a specific increase in Hif2a (Epas1) transcription and its target genes in GPe PV+ neurons. The animals are referred to as S1 and S2 for sham animals and DD1-4 for 6-OHDA-treated animals in order of depletion severity. TH levels are shown for each animal in the bar graph. D, There is an increase in the proportion of GPe PV+ cells that express Hif2a protein between images from healthy tissue and DD tissue (t test) (Extended Data Figure 4-4).