Fig 2: HIP knockdown activates canonical Hh target genes and enhances Hh responsiveness. (A) Inhibition of HIP increased the expression levels of Gli-1 and VEGFA. (B) eEPCs, LEPCs, and LEPCs with HIP knockdown were treated for 12 h with 100 ng/ml Shh, and then Gli-1 activation was assessed using qRT-PCR. (C,D) LEPCs and LEPCs with low HIP expression were treated with vehicle (DMSO), Hh inhibitor cyclopamine (20 µM) or Gli-1-specific inhibitor GANT61 (20 µM) for 12 h, and then Gli-1 expression was measured. (E) After treatment with 20 µM GANT61 for 24 hours, LEPCs expressing either scrambled or HIP shRNA were detached and seeded on Matrigel with 10 ng/ml VEGF. After 12 h, the tubes were counted. (F) After treatment with 20 µM GANT61 for 24 hours, LEPCs expressing either scrambled or HIP shRNA were treated with 50 µM H2O2 for 3 h, followed by the MTT assay.

Fig 3: Shh significantly activates Hedgehog signaling via the canonical Gli-1-dependent pathway in LEPCs. (A–F) LEPCs were cultured with 100 ng/ml Shh, and then canonical Hh target genes, including Gli-1, Ptc-1, VEGFA, and ANG1, and non-canonical Hh signal target genes, including OPN and MMP2, were analyzed using qRT-PCR. (G–J) HUVECs were cultured with 100 ng/ml Shh, and then the expression levels of Gli-1, OPN and MMP2 were analyzed. (K) LEPCs and HUVECs were cultured with 0–100 ng/ml Shh for 12 h, and then Gli-1 expression was assessed using Western blot analysis. (L–N) LEPCs and HUVECs were incubated with 200 ng/ml Shh and 20 ng/ml VEGF for 12 hours on matrigel and tube formation was observed under light microscope (scale bar, 100 µm).