Fig. 4

Age-dependent decrease in mTORC2 contributes to pro-inflammatory phenotype. a Protein analysis using the Wes instrument (Protein Simple) analyzing levels of RICTOR in primary microglia. b Graph representing measurements from RICTOR western show aged microglia have diminished levels of the RICTOR protein (^## p < 0.01 Student’s unpaired t test, N = 3). c Primary microglia young (5–7 months) and old (20–24 months) were treated with 100 ng/ml IGF for 15 min and then protein samples analyzed for AKT and AKTp473. d Analysis of intensity measurements received from the WES indicates pAKT activation by IGF is lower in aged microglia (two-way ANOVA, there was a significant effect of age F(1,8) = 60.8, ^## p < 0.01 and a significant effect of treatment F(1,8) 33.45 ^## p < 0.01. N = 3). e PCR analysis of primary microglia shows increased pro-inflammatory gene expression at baseline; values are expressed as fold change from young. f Primary microglia were stimulated with 20 ng/ml TNFα for 24 h. g Aged microglia have higher change from their respective baseline than young microglia for TNF, IL1β, IL6, and Marco. h Primary microglia were stimulated with IL4 20 ng/ml for 24 h. A reduced change (>−1.5-fold) from baseline in the aged compared to control was observed for Arg1 and FIZZ1. i BV2 cells (siRNA Rictor 48 h) treated with TNFα had higher levels of inflammatory gene expression IL1β and IL6 than a scramble control group. j BV2 cells pretreated with siRNA and scramble RNA for 48 h and subsequently treated with IL4 (20 ng/ml) for 24 h had lower expression of genes Arg1 and Fizz1 associated with resolution of the inflammatory response. Fold changes for baseline that are reported are relative comparisons using Young/Scramble as control. Interpreted as had lower expression of anti-inflammatory genes as compared to young microglia. N = 3 biological replicates per experiment examined in triplicate. For the TNFα and IL4 stimulation experiments, data is represented as the ratio of fold change from age/treatment matched control. Interpreted as the fold change from baseline in aged microglia for IL6 in response to TNFα is sevenfold higher than that observed in young microglia. Fold change of greater than 1.5 is considered significant (marked by number sign), all changes are illustrated, not just those that met criterion