Fig 2: Characterization of Arnt SMKO mice. Isolated aortic vascular smooth muscle cells from Arnt SMKO mice show loss of aryl hydrocarbon receptor nuclear translocator (ARNT) expression in mRNA (n=5; A) and total protein (n=3; B). Tissue samples from gastrocnemius muscle show no differences in bulk ARNT expression in mRNA (n=5; C) or total protein (n=4; D). E, Body weights of male mice are similar in Arnt SMKO (n=8) and Arnt lox/lox (n=11) littermates. Smooth muscle–specific ARNT deletion does not compromise cardiac function assessed by heart rate (F), ejection fraction (G), fractional shortening (H), and left ventricular (LV) mass (I) on echocardiogram (n=5). Unpaired 2-tailed t test (A through F, H, and I) with Mann-Whitney test (G) or Welch's correction (H) was used. *P<0.05.

Fig 3: Assessment of vascular smooth muscle cell (VSMC) phenotype in vitro. A, Migration in normoxic (21% O2) or hypoxic (2% O2) conditions was detected in a 2-dimensional scratch assay measured after 6.5 hours. Arnt SMKO VSMCs show decreased migration in hypoxia compared with Arnt lox/lox cultures (n=9). B, Quantitative analyses of immunopositive 5-bromo-2'-deoxyuridine (BrdU) cells reveal increased proliferation of Arnt SMKO VSMCs in normoxia and hypoxia (n=3). 1- way ANOVAs were performed. DAPI indicates 4',6-diamidino-2-phenylindole. *P<0.05, Arnt SMKO vs Arnt lox/lox; ^ P<0.05, hypoxia vs normoxia.

Fig 4: Histological assessment of capillary number and perfusion status in gastrocnemius muscle (GC). A, Representative micrographs of perfused vessels assessed by staining sections for CD31 (green) from GCs processed after mice were infused with endothelium-binding intravenous lectin at day 7 after femoral artery ligation (red). Although the number of CD31+ capillaries per field at day 7 is not significantly different (B), ligated limbs of Arnt SMKO and Arnt lox/lox mice show decreased CD31+ capillaries/myocyte (C) and fraction of perfused double-positive vessels (D) compared with unligated limbs at day 7 (n=4). No significant differences were seen between Arnt SMKO and Arnt lox/lox in any of the above metrics at day 7. E, Representative images of immunostained sections for CD31-labeled vessels (green) and a-smooth muscle actin (SMA; smooth muscle cells; red) from day 28 after ligation of GCs. F, Numbers of CD31+ capillaries per field in day 28 ligated limbs of Arnt SMKO and Arnt lox/lox are similar to, if not increased, over unligated limbs (n=3). G, Ligated limbs of Arnt SMKO mice show decreased CD31+ capillaries/myocyte compared with ligated limbs of Arnt lox/lox mice at day 28 (n=3). Scale bar=50 µm. H, Day 28 ligated limbs of Arnt SMKO and Arnt lox/lox have similar numbers of CD31+ SMA + vessels (n=3). One-way ANOVA (B), Kruskal-Wallis test (C, D, F, and G), or unpaired 2-tailed t test (H) was used. D indicates day. *P<0.05, Arnt SMKO vs Arnt lox/lox; ^ P<0.05, ligated vs unligated.

Fig 5: HIF-1ß is required for viability of cells and Drosophila melanogaster following infection. (A) HeLa cells were transfected with control or HIF-1ß siRNA oligonucleotides. Where indicated, cells were treated with 100 ng/mL LIGHT for 4 or 24 h prior to whole cell lysis and Western blot analysis. Actin was used as a loading control. (B) HeLa cells were transfected as in 4A, but treated with 10 n/mL TNF-a for 4 h prior to lysis and Western blot analysis. Actin was used as a loading control. (C) Wild-type adult flies (w1118) and HIF-1ß (Tango) loss-of-function flies (Tango LF) were pricked using a thin needle dipped in a diluted overnight culture of Serratia marcescens Db10 (OD600 = 0.2) or in a saline solution (PBS, Mock). Groups of 100 flies were used and kept at room temperature. Survival was monitored and expressed as the ‘estimated probability of survival’. The p-value was obtained from log-rank statistical analysis: * p < 0.05. (D) Wild-type adult flies (w1118) and HIF-1ß (Tango) loss-of-function flies (Tango LF) were pricked using a thin needle dipped in a culture of Escherichia coli or in a saline solution (PBS, Mock). RNA was extracted 2 h later. Following cDNA synthesis, qPCR was performed using the indicated primers. Graphs depict mean and SEM from three independent biological experiments. Student t-test analysis was performed for each gene and levels of significance determined as: ns = not significant, * p < 0.05; *** p < 0.001.