Fig. 3

P4HA1 knockdown suppresses angiogenesis in vitro and in vivo. A Validation of knockdown efficiency of Ad-shP4HA1 in HUVECs (n = 3). B–D After HUVECs were infected with Ad-shNC and Ad-shP4HA1 for 24 h, B EdU incorporation assay (magnification: ×100, scale bar: 100 μm), C tube formation assay (magnification: ×100, scale bar: 100 μm), and D transwell migration assay (magnification: ×400, scale bar: 50 μm) were performed (n = 3). E In vivo, P4HA1 knockdown was achieved by locally injecting purified adenoviruse into the gastrocnemius muscle on the first and seventh day after surgery. The knockdown efficiency of P4HA1 in gastrocnemius muscle of the HLI model (14 d) was detected and analyzed (n = 5). F Representative Doppler images of blood flow recovery after HLI with P4HA1 knockdown. G Statistical analyses of blood flow recovery in the HLI model with P4HA1 knockdown (n = 5). H Immunofluorescence staining of CD31 (green) in the gastrocnemius muscle from HLI model (14 d) with P4HA1 knockdown. Magnification: ×400, scale bar: 50 μm. The fluorescence intensity was statistically analyzed (n = 5). Data in A–E, H were analyzed using the Student’s t-test. Data in G were analyzed by two-way ANOVA with Bonferroni post hoc test. *p < 0.05. NC negative control, HLI hindlimb ischemia