Fig 2: 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.

Fig 3: Bulk perfusion and functional recovery are reduced in Arnt SMKO mice after femoral artery ligation. Ligations distal to the deep femoral artery branch point were performed in the left limbs of age- and sex-matched adult Arnt SMKO and Arnt lox/lox mice. A, Representative images of perfusion measured using infrared laser Doppler scanning for Arnt SMKO and Arnt lox/lox mice over 21-day recovery. B, Perfusion, reported as a ratio of ligated/unligated limb, was significantly lower in Arnt SMKO compared with Arnt lox/lox mice from days 7 through 21 (n=10, repeated-measures ANOVA). C, Functional scoring on days 3 and 7 shows impairment trend in functional recovery in Arnt SMKO mice (n=11). D indicates day; Pre-op, preoperatively; Post-op, postoperatively. *P<0.05, Arnt SMKO vs Arnt lox/lox.

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: Smooth muscle morphologic features and perimural wrapping of small arterioles. A, Representative confocal images of smooth muscle actin positive (SMA+; cyan) vascular smooth muscle cells (VSMCs) around small arterioles in spinotrapezius muscle illustrate disruption of organization and VSMC morphologic features in Arnt SMKO mice (n=3). Top row scale bar=60 µm. Bottom row scale bar=10 µm. B, Representative peripheral arterioles in gastrocnemius muscles of Arnt SMKO mice also display aberrant morphologic features and organization of SMA + VSMCs visualized by multiphoton microscopy (n=3). Scale bar=60 µm; insets, ×3 magnification. D indicates day.