Fig. 2: Shear-stress-induced PI3P synthesis at the PC depends on PI3KC2α.
a Representative confocal images upon shear-stress conditions of PI3KC2α knocked down HK2 cells (siPI3KC2α), compared to control cells (siCTRL), immunostained for ARL13B, PI3P (using FYVE-GST indirect recombinant peptide) and DAPI (N = 80 cells, from five independent experiments). Scale bar, 10 μm. b Schematic drawing showing the 150 µm2 circular area centred at the cell nucleus used as identifier of the PC basal body area. c Quantification of the PI3P-positive structures at the PC area, as defined in (b) and shown in (a), in siCTRL or siPI3KC2α HK2 cells, upon static (ctrl) and shear-stress (96 h) conditions (mean ± SEM, N = 80 cells, from five independent experiments). NS: not significant, ***p < 0.001 in two-tailed Student’s t test. d Western blot analysis and quantification of WIPI2 protein levels in lysates of polarized siCTRL or siPI3KC2α HK2 cells, upon static (ctrl) and shear-stress (96 h) conditions. Bar graph denotes average protein levels normalized to actin (mean ± SEM, from three independent experiments). NS: not significant, ***p < 0.001 in two-tailed Student’s t test. e WIPI2 mRNA levels quantifications from total lysates of polarized siCTRL or siPI3KC2α HK2 cells, upon static (ctrl) and shear-stress (96 h) conditions. Bar graphs denote fold change of average mRNA levels relative to ctrl and normalized to actin mRNA (mean ± SEM, from three independent experiments). ***p < 0.001 in two-tailed Student’s t test. f Representative confocal acquisition of WIPI2GFP transfected HK2 cells upon shear-stress conditions immunostained for ARL13B, PI3P (using FYVE-GST indirect recombinant peptide) and DAPI showing PI3P and WIPI2GFP colocalization at the base of PC (arrowhead). Scale bar, 10 μm.
