Fig. 4. Exogenous PI3P delivery restores autophagy and cell size regulation upon shear stress in PC-deprived cells.

a Schematic display of the experimental design; all cells were analyzed after 96 h of fluid flow. We added to IFT88 knockdown (siIFT88) HK2 cells cells two different concentrations of exogenous PI3P (DiCI6.PI3P, in lipid carrier vehicle, at 0.1 and 1 μM) during the last 3 h of fluid-flow treatment and compared them to control cells (siCTRL) and to siIFT88 cells added with carrier alone, as a negative control. b Western blot analysis of IFT88 in lysates of HK2 cells (siCTRL) and IFT88 knocked down cells (siIFT88). c, e Representative confocal images of HK2 cells (siCTRL), compared to siIFT88, supplemented with exogenous PI3P (0.1 and 1 μM) or with carrier only, upon shear-stress (96 h) conditions, immunostained for LC3 and DAPI or F-actin and DAPI, respectively. Scale bar, 10 μm. d, f Quantifications of c and e. Bar graphs denote average number of LC3 puncta per 200 µm² area and average cell surface, respectively (mean ± SEM, N = 70, from three independent experiments). **p < 0.01, ***p < 0.001 in two-tailed Student’s t test.