Fig. 5

PI3K-C2α and PI3K-C2β are required for clathrin-mediated pinocytosis. a Effects of double knockdown of PI3K-C2α and -C2β on FITC-dextran uptake. Cells were transfected with scrambled, PI3K-C2α or -C2β siRNAs (50 nM each) or with the combination of C2α- and C2β-siRNAs (25 nM each). Left, Relative protein expression levels of PI3K-C2α and -C2β determined by western blotting. Right, FITC-dextran uptake, b No effect of C2α- and C2β-knockdown in CHC-depleted HUVECs. The single and double knockdown was performed as described in the Materials and Methods. After 48 h, cells were subjected to the FITC-dextran uptake assay as described in a. c, d Super-resolution microscopic images of cells transfected with the expression vectors of mRFP-clathrin light chain (mRFP-CLC, red) and either GFP-C2α (green in c) or GFP-C2β (green in d). Nuclei were stained with DAPI (blue). Scale bar: 10 μm. Lower, Representative serial images showing the dynamics of PI3K-C2α (c) and -C2β (d) association with clathrin. Time zero was set as the peak of clathrin recruitment. See also Electronic Supplementary Material movies 1 and 2 with movie legends. e Quantified data of the lower images in c and d. n = 3 and 4 events for C2α/clathrin and C2β/clathrin, respectively. f Co-localization of GFP-C2α (left) or -C2β (right) in clathrin-coated pinocytic vesicles. Super-resolution microscopic images of cells expressing GFP-C2α (left) or -C2β (right) and mRFP-CLC that were subjected to Alexa Fluor 647-dextran uptake. White arrowheads indicate dextran-containing C2α- or C2β-positive pinosomes. g Effects of either PI3K-C2α or -C2β knockdown on FITC-dextran accumulation in EE. Cells were transfected with scrambled, PI3K-C2α or -C2β siRNAs. 24 h later, the knocked down cells were infected with RFP-Rab5 baculoviral vector, followed by FITC-dextran uptake assay. Left, representative images and right, quantified data of FITC-dextran⁺, mRFP-Rab5⁺ vesicles and mRFP-Rab5⁺ vesicles. h Time-dependent degradation of uptaked FITC-dextran in PI3K-C2α- and -C2β-depleted cells. Cells that had been transfected with indicated siRNAs were subjected to FITC-dextran pulse-chase experiments as described in the Materials and methods. Nuclei were stained with DAPI (blue). Data are presented as the median and IQR in a, right, b and g and as the mean ± SEM in a, left, e and h. Asterisks indicate statistically significant difference between the indicated groups: *p < 0.05, **p  < 0.01, ***p < 0.001; ns not significant