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[Preprint]. 2024 Apr 1:2024.04.01.587559. [Version 2] doi: 10.1101/2024.04.01.587559

Figure 2: Impaired proteasomal/lysosomal function leads to increased angiogenesis in vitro and in vivo.

Figure 2:

A. Schematic of the in vitro 3D collagen type I invasion/angiogenesis assay. HUVECs are transfected with either DYNC1LI1 or Control siRNA and seeded onto a collagen type 1 gel. After 24 hours, assays are observed for cellular invasion from the monolayer. B, C. Representative cross-sectional images of 3D collagen invasion assays, showing an increase in the number of invading cells in siDYNC1LI1-transfected HUVECs (B) compared to siControl-transfected cells (C). D. Quantification of the average number of invading cells in siControl versus siDYNC1LI1 conditions. E. Western blot analysis shows decreased expression of LIC1 in siDYNC1LI1-treated HUVECs compared to siControl HUVECs. Alpha-Tubulin is shown as a loading control. F. Quantification of the average number of invading HUVECs in 3D collagen gel assays. Assays were treated with inhibitors that block discrete stages of endocytosis. Dynasore (10uM), an early endosomal inhibitor, inhibited HUVEC invasion into the collagen gels. Bortezomib (100nM) and MG132 (10uM), which are late endosome/lysosome inhibitors, increased HUVEC invasion, consistent with siDYNC1LI1 results. G. Confocal images of 96 hpf zebrafish embryos treated with MG132 (10nM) or DMSO vehicle control starting at 4 hpf to investigate the effects of late endosome/lysosome inhibition on angiogenesis in vivo. WT Tg(fli:eGFP) zebrafish were used for these studies. MG132-treated fish phenocopy the dync1li1y151/y151 mutant over-branching phenotype. H. Percentage of ISVs displaying ectopic sprouts in DMSO- or MG132-treated embryos at 96 hpf. n=20–33 embryos. I. Schematic of LIC1’s proposed role in regulation of endocytosis. For panels D and F, each dot represents an individual 3D collagen assay. Statistics for panel D were calculated using a Mann-Whitney test. Statistics for panel F were calculated using one-way ANOVA with Dunnett’s multiple comparisons test; omnibus ANOVA P-value for F (prior to the post hoc tests) is <0.0001. Statistics for panel H were calculated using an unpaired t-test with Welch’s correction. Data are presented as the mean ± S.D. Scale bars: 100um (B) and 50um (G).