Fig 2: Modulation of PI3K-Akt pathway affects autophagic and phagocytic activity as well as inflammatory response in microglial cells. a A representative Western blot image of lysates of BV2 cells treated with LY294002 (LY) (0.1 µM – 10 µM), and b with LY294002 together with LPS. c Quantification revealed that LY294002 decreased Akt S473 phosphorylation levels dose-dependently in cells treated with LY294002 and together with LPS and LY294002. d Quantification of LC3B II and I levels revealed a significant increase in LCB3II/I ratio by LY294002. e Phagocytic activity in BV2 cells was assessed using pHrodo bioparticles and fluorescence emission was measured in the IncuCyte live cell imaging device in every 15 min. BV2 cells were treated with four different concentrations of PI3K inhibitor LY294002 (5 µM – 50 µM) and with 5 µM Cytochalasin D (CytD). LY294002 decreased phagocytic activity dose-dependently (p < 0.001) and CytD blocked phagocytosis almost completely. a-d; Data are presented as mean + SEM from four biological replicates. Mann-Whitney U-test, *p < 0.05, **p < 0.01, ***p < 0.001 vs. untreated control, #p < 0.05, ##p < 0.01, ###p < 0.001 vs. LPS control. f Phagocytic activity of primary microglia isolated from WT and Akt2 KO mice was assessed similarly as in BV2 cells using pHrodo bioparticles and fluorescence emission was measured in the IncuCyte live cell imaging device in every 30 min. Cells were treated with three different concentrations of LY294002 (1 µM – 25 µM) and with 5 µM CytD. LY294002 decreased phagocytic activity dose-dependently (p < 0.001) and Akt2 KO cells showed significantly decreased phagocytic activity as compared to WT cells (p < 0.001). CytD blocked phagocytosis almost completely in both WT and Akt2 KO cells. e-f; Data are presented as mean + SEM from three biological replicates in technical quadruplets, One-way ANOVA, LSD (e) and Two-way ANOVA (f). g Levels of inflammatory markers, nitric oxide (NO), tumor necrosis factor a (Tnfa), and interleukin 6 (Il6) were significantly lower in Akt2 KO primary microglia as compared to WT primary microglia after 48 h LPS + IFN? -treatment. Data presented as mean % + SEM, n = 3–4. *p < 0.05, **p < 0.01, T-test