Journal: The Journal of Biological Chemistry

Article Title: Akt3 kinase suppresses pinocytosis of low-density lipoprotein by macrophages via a novel WNK/SGK1/Cdc42 protein pathway

doi: 10.1074/jbc.M116.773739

Figure Lengend Snippet: Increased activity of SGK1 promotes pinocytosis in Akt3−/− macrophages. A, phosphorylation of Akt (Ser-473/Thr-308), total Akt, and Akt1/2 expression in WT and Akt3−/− MPMs treated with 50 ng/ml M-CSF. Ctl, control without M-CSF treatment. n = 3. B, uptake of Lucifer yellow dye by WT and Akt3−/− MPMs in the presence of 10 μm GSK3 inhibitor (SB), 20 nm mTOR inhibitor (RAPA), or 2 μm NFκB inhibitor (BAY). n = 8. C, phosphorylation and expression of NDRG1, an SGK1 specific substrate, in WT and Akt3−/− MPMs assessed by Western blotting. Bottom panel, expression of SGK1 in WT and Akt3−/− MPMs. n = 3. D, Lucifer yellow uptake by MPMs in the presence of SGK1 inhibitor (K-650394). n = 8. E, foam cell formation assay in WT and Akt3−/− MPMs cultured in the presence of 1 mg/ml LDL and in the presence or absence of 25 μg/ml SGK1 inhibitor (SGK1i) for 24 h (n = 8). F, effect of SGK1 inhibitor (GSK-650394, 25 μg/ml) on the uptake of Lucifer yellow by WT and Akt3−/− MPMs (n = 7). G, effect of Akt3 siRNA treatment on phosphorylation of NDRG1 and expression of NDRG1, Akt3, and Cdc42 in human MDMs assessed by Western blotting. GAPDH expression was used as a loading control (n = 6). H, uptake of Lucifer yellow dye by human MDM treated with control siRNA or Akt3 siRNA in the presence of 25 μg/ml SGK1i (n = 6). I, effect of 25 μg/ml SGKi on cholesterol accumulation in human MDM (n = 6). Human MDM were treated with Akt3 or control siRNA treatment and then cultured in the presence of 1 mg/ml LDL and 25 μg/ml SGK1i overnight, and cholesterol content was quantified. Data represent means ± S.E. *, p < 0.05. Data are representative of at least three independent experiments and are quantified from at least three independent experiments.

Article Snippet: Cdc42 activity in cell lysates was measured using a RhoA/Rac1/Cdc42 Activation Assay Combo Biochem Kit (Cytoskeleton) ( n = 3).

Techniques: Activity Assay, Expressing, Control, Western Blot, Tube Formation Assay, Cell Culture

Journal: The Journal of Biological Chemistry

Article Title: Akt3 kinase suppresses pinocytosis of low-density lipoprotein by macrophages via a novel WNK/SGK1/Cdc42 protein pathway

doi: 10.1074/jbc.M116.773739

Figure Lengend Snippet: Akt3 inhibits macrophage pinocytosis via the SGK1/Cdc42 pathway. A, uptake of Lucifer yellow by WT and Akt3−/− MPMs in the presence or absence of 10 μm Cdc42 inhibitor (ML141). n = 8. B, uptake of Lucifer yellow dye by human MDMs treated with control siRNA or Akt3 siRNA in the presence of 10 μm ML141 (n = 6). C, cellular cholesterol levels of human MDMs treated with Akt3 siRNA or control siRNA in the presence of 1 mg/ml LDL and 10 μm ML141 (n = 6). D, Cdc42 activity in WT and Akt3−/− MPMs. Macrophages were treated with 25 μg/ml SGK1i overnight. Cells were then washed and lysed. Cdc42 activity in cell lysates was measured using a RhoA/Rac1/Cdc42 Activation Assay Combo Biochem Kit (Cytoskeleton) (n = 3). E, expression of Cdc42 in WT and Akt3−/− MPMs cultured in the presence or absence of 25 μg/ml SGK1i or 1 mg/ml LDL (n = 3). F, effect of suppression of Akt3 expression (siRNA treatment for 48 h, Akt3i) on Cdc42 expression in human MDM as assessed by Western blot analysis. GAPDH expression was used as a loading control (n = 6). G, expression of Rac1 in macrophages treated with 25 μg/ml SGK1 inhibitor and 0.8 mg/ml LDL (top panel). (n = 3). H, effect of SGK1i on RhoA expression in WT and Akt3−/− MPMs. Cells were incubated with or without 25 μg/ml SGK1i overnight. Cell protein samples were assessed by Western blot analysis. I, F-actin formation in WT and Akt3−/− MPMs exposed to 500 μ/ml LDL in the presence or absence (control) of SGK1 inhibitor. Center panels, F-actin formation (white arrows) at higher magnification. The actin cytoskeleton and the nuclei were visualized by staining with Alexa Fluor 488-phalloidin and DAPI, respectively. Scale bars = 25 μm, n = 8. Right panels, quantification of phalloidin fluorescence intensity. J, macrophages treated with 500 μg/ml LDL in the presence or absence of 25 μg/ml SGK inhibitor (GSK650394) for 4 h. Cells were stained with 0.6 μm TRITC-phalloidin. TRITC-phalloidin·F-actin complexes were extracted, and fluorescence was measured by SPECTRAmax GEMINI XS. Data represent means ± S.E. *, p < 0.05. Data are representative of at least three independent experiments.

Article Snippet: Cdc42 activity in cell lysates was measured using a RhoA/Rac1/Cdc42 Activation Assay Combo Biochem Kit (Cytoskeleton) ( n = 3).

Techniques: Control, Activity Assay, Activation Assay, Expressing, Cell Culture, Western Blot, Incubation, Staining, Fluorescence