Fig 2: (A) Western blot analysis of kidney homogenate pSGK1 following the RI infusion of the following (n = 6 for each condition): vehicle control D5W, ghrelin (3 μg/min), ghrelin + PKA inhibitor (iPKA) Rp-cAMPS (10 μg/kg/min), and ghrelin + PI3K inhibitor (iPI3K) LY (0.1 μg/kg/min). (B) Western blot analysis of kidney homogenate total SGK for the same four conditions in A. (C) Ratio of pSGK1/total SGK (each normalized to total protein) for the same four conditions in A. (A and B) Arrows on the left indicate pSGK1 and SGK, respectively, whereas arrows on the right depict the molecular weight ladder. Data represent means ± 1 SE. *P < 0.05, **P < 0.01, and ***P < 0.001 from control. Overall ANOVA analyses: (A) F = 21.2, P < 0.0001; (C) F = 11.4, P < 0.0001.

Fig 3: Effects of polyphenols and rapamycin on the protein expression and phosphorylation of serum- and glucocorticoid-regulated kinase 1 (SGK1) in prostate cancer cells. DU-145 and PC-3 cells were cultured with vehicle (DMSO), the indicated concentrations of curcumin (CUR) and carnosic acid (CA), alone and in combination, or the mTOR inhibitor rapamycin, for 6 or 24 h. Cells samples were then subjected to Western blot analysis. Integrated Density Values (IDVs) of the indicated protein bands normalized to IDVs of respective GAPDH bands are shown below corresponding blot images. Representative blots of 3 similar experiments are presented.

Fig 4: Effects of polyphenols and rapamycin on mitochondrial respiration and expression of mTOR downstream genes. (a) Rapamycin inhibits the effects of the polyphenol combination on mitochondrial respiration. Cells were pretreated with vehicle (DMSO) or 5 μM rapamycin (Rap) for 1 h followed by the addition of vehicle or 7 μM CUR and 5 μM CA (CUR + CA). Data are presented as mean ± SEM (n = 3) relative to DMSO. Statistically significant differences for the indicated groups vs. DMSO (* p < 0.05 and ** p < 0.01). § p < 0.05, significant differences between the indicated groups (Student’s t-test). (b) Changes in mTOR downstream transcriptional profile upon treatment of DU145 cells with 5 μM rapamycin or 7 μM CUR, 5 μM CA or CUR + CA) for 24 h, relative to DMSO. (c) Quantitative analysis of SGK1 gene expression. Data are presented as fold regulation vs. DMSO (mean ± SEM; n = 3), where DMSO is set as 0. Statistically significant differences for treatments vs. control (DMSO): * p < 0.05 and ** p < 0.01.

Fig 5: (A) Western blot analysis of pSGK1 in response to the RI infusion of the following (n = 6 for each condition): vehicle control D5W, ghrelin (3 μg/min), ghrelin + microtubule inhibitor NOC (3 μg/kg/min), and ghrelin + actin inhibitor CTD (0.3 μg/kg/min). (B) Western blot analysis of total SGK for the same four conditions in A. (C) Ratio of pSGK1/total SGK (each normalized to total protein) for the same four conditions in A. (A and B) Arrows on the left indicate pSGK1 and SGK, respectively, whereas arrows on the right depict the molecular weight ladder. Data represent means ± 1 SE. **P < 0.01 and ***P < 0.001 from control. Overall ANOVA analyses: (A) F = 14.9, P < 0.0001; (C) F = 13.1, P < 0.0001.