Fig. 3
QKI loss mediated up-regulation of autophagy is dependent on PI3K-p110β. A, B Immunoblot analysis of protein expression of Vps15, Vps34, Beclin-1, PI3Kp110α, PI3K-p110β and PI3Kp110δ after SC and shQKI5 RAW264.7 cells infected with MRSA for indicated durations. Actin was an internal control. One representative immunoblot is shown (A) and the graph (B) presents quantification of the band intensity for immunoblots from three independent experiments. C–D Immunoblot analysis of PI3Kp110α, PI3K-p110β, PI3K, AKT, phospho-AKT (Ser473), mTOR, and phospho-mTOR (Ser2448) protein levels in lysates of bone marrow-derived macrophages (BMDM) from QKIfl/fl and LysM+QKIfl/fl mice stimulated with MRSA infection. Actin was an internal control. One representative immunoblot is shown (C) and the graph (D) presents quantification of the band intensity for immunoblots from three independent experiments. E Western blots were performed to determine the effects of inhibitor of PI3K-p110β “TGX-221” after MRSA infection 4 h. Total cell lysates were subjected to western blot to analyze the protein expression of LC3-II, with actin as an internal control. One representative immunoblot is shown (E). Graph (F) are representative quantification of the band intensity for immunoblots from three independent experiments. The results were shown as expression of LC3-II. F Immunofluorescence showed the expression of LC3-II in QKI silenced (ShQKI) and scramble (SC) RAW264.7 cells treated with TGX-221 under infection conditions. LC3-II was labeled in red and DAPI staining was shown in blue (Scale Bar: 10 μm). The mean fluorescence intensity of LC3-II immunostaining was showed on the right. G “Phagocytosis assay” was performed to determine the effect of TGX-221.The number of bacteria units forming were calculated after 8 h. Enumeration of forming bacteria units (c.f.u) were shown (right). Representative graph of bacteria forming units were showed in the left. H Representative imagines showed the bacteria forming units after LysM+QKIfl/fl mice were treated with inhibitor of PI3K-p110β “TGX-221”. Enumeration of forming bacteria units (c.f.u) from various organs (kidney, spleen, liver) were analyzed I Cytokines (TNF-α and IL-1β) level in blood were analyzed by ELISA method after LysM+QKIfl/fl mice were infected 24 h and treated with inhibitor of PI3K-p110β “TGX-221”. All the bars represented the mean of measurements from three independent experiments, and the error bars indicated ± SD. (A-E) are representative of three experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (student’s t test). H, one-way ANOVA with Tukey’s multiple comparisons test. G are representative of one experiment (n = 3 wells/group). H, I are from one experiment (H, n = 8 mice/group I, n = 4 mice/group). Symbols represent data points for each animal. *p < 0.05, **p < 0.01, ***p < 0.001, not significant (ns) (student’s t test)