Fig. 1

Class II phosphoinositide 3-kinase (PI3K)-C2α and PI3K-C2β are necessary for fluid-phase pinocytosis in human umbilical vein endothelial cells (HUVECs). a, b Time-dependent uptake of fluorescein isothiocyanate (FITC)–dextran. Cells were incubated with FITC-dextran (0.2 mg/ml) in growth medium at 37 °C for the indicated time periods, followed by fixation with 4% paraformaldehyde. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Scale bar: 20 μm. a Representative images showing internalized FITC-dextran (green dots) at each time point. b Quantified data (n = 50–60 cells were analyzed in each group). The green dots were quantified as described in the Materials and methods. c Small interfering RNA (siRNA)-mediated knockdown of PI3K isoforms. Representative western blots (WB) showing siRNA-mediated knockdown of PI3K protein expression after 48 h post-transfection (left). Cells were transfected with either PI3K-C2α-, PI3K-C2β-, p110 α- or Vps34-specific siRNA or scrambled siRNA. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading control. Quantified data of protein expression levels (right). d, e Effects of knockdown of different PI3K isoforms on FITC-dextran uptake. Cells were incubated with FITC-dextran for 45 min. d representative images, e quantified data. f Effects of different PI3K inhibitors on FITC-dextran uptake. Cells were pretreated with the inhibitors GDC-0941 (0.1 µM), 3-methyladenine (3-MA; 1 mM) or wortmannin (WMN; 30 nM and 1 µM) for 30 min and then subjected to the FITC-dextran uptake assay. Scale bar: 20 μm. Data are presented as the mean ±  standard error of the mean (SEM) in  c and as the median and interquartile range (IQR) in b, e and f. Asterisks indicate statistical significance between the indicated groups: *p < 0.05, **p < 0.01, ***p < 0.001