Fig 2: PI3K-C2α is required for clathrin-dependent endocytosis of γ-secretase complex. (a, b) The effects of PI3K-C2α (a) and CHC (b) knockdown on the cell surface Nicastrin (Nct) level in HUVECs. siRNA-transfected cells were incubated on Dll4- or BSA-coated plates for 24 h. Total cell lysate and biotin-labeled surface proteins were subjected to Western blot analysis. (n = 5). (c) The effects of CHC knockdown on Notch1 localization in HUVECs. CHC-siRNA-transfected cells were incubated on Dll4- or BSA-coated dishes for 24 h. Note the accumulation of Notch1 at perinuclear vesicles (yellow arrowheads) in CHC-depleted cells (#), but not in CHC-expressing cells (*). (d) Expression of Nct-GFP protein in HEK293T cells. Cells were transfected with Nct-GFP expression vector (2 μg) or empty vector (pAcGFP1-N, 2 μg). 12 h after transfection, cells were harvested for Western blot analysis for Nct (upper panel) and GFP (lower panel). (e) The effects of PI3K-C2α knockdown on the localization of Nct-GFP in HUVECs. Cells were transfected with Nct-GFP expression vector (2 μg) and either PI3K-C2α-siRNA or ctrl-siRNA, and incubated on Dll4- or BSA-coated dishes for 2 h or 4 h. The SRRF views of the boxed regions in confocal views are also shown in the right upper and lower of each panel. Note the intracellular colocalization of Nct-GFP and Notch1 (white arrowheads) at LAMP1-positive lysosomes (white arrows), which was more abundant in control cells compared to PI3K-C2α-depleted cells. Nct-GFP was also accumulated at the cell surface of PI3K-C2α-depleted cells (yellow arrowheads). Dashed lines in the SRRF views (right lower of each panel) delineate the nuclei. (f) Quantification of colocalization efficiency of Nct-GFP and Notch1. The colocalization index was quantified using Image-J software. Pearson’s R-value is shown (n = 5–8). (g) The effects of PI3K-C2α knockdown on Dll4-induced expression of EndMT-related genes in HUVECs. PI3K-C2α-siRNA- or ctrl-siRNA-transfected cells were incubated on Dll4- or BSA-coated plates and 24 h later harvested for qPCR analysis (n = 5). Statistical significance was assessed with two-way ANOVA followed by Bonfferroni’s post-hoc test in (a, b, f, g). Statistical significance was presented as *P < 0.05, **P < 0.01 or ***P < 0.001, respectively. ns: not significant.