Fig. 6

MTOR and RPS6 transcriptomic and western blot analyses with RNAs and protein extracts from L. m.-infected and HBSS-starved BMDM, and determination of infection rates of L. m.-infected BMDM after MTOR downregulation by RNA interference. Methods: (a, b) BMDM from BALB/c mice were infected with L. m. promastigotes for 1 or 24 h. Uninfected control BMDM were incubated for the same amount of time in RPMI medium or starved for 1 h in HBSS. Proteins were harvested and subjected to western blot analyses with specific antibodies against MTOR, p-MTOR, RPS6, and p-RPS6. Western blots with proteins from 3 independent experiments were analyzed densitometrically. ACTB served as the internal loading control. Total RNA was harvested from L. m.-infected BMDM and uninfected control BMDM. The Affymetrix® chips were hybridized with RNA samples from 2 independent experiments were analyzed densitometrically. c Additionally, BMDM were transfected with specific siRNA 4 h prior to infection to downregulate the expression of MTOR, and the cells were finally infected with L. m. promastigotes. L. m.-infected controls were transfected with negative control siRNA. The infection rates were determined 48 h p.i. in 2 independent experiments. Results: (a) A significant hyperphosphorylation was observed for MTOR and RPS6 in samples from L. m.-infected BMDM 1 h p.i. compared to uninfected control BMDM. b Results of densitometric analyses of western blot experiments and Affymetrix® chip analyses showed that MTOR and RPS6 expressions were not regulated at the mRNA or the protein level. However, MTOR and RPS6 were significantly hyperphosphorylated in L. m.-infected BMDM 1 h p.i.. c A significant decrease in the infection rate was detected in L. m.-infected BMDM after downregulation of the protein expression of MTOR compared to L. m.-infected BMDM transfected with negative control siRNA. L. m.-inf. = L. m.-infected, neg. control = negative control, n.s. = not significant, RFU = relative fluorescence units, * p ≤ 0.05, ** p ≤ 0.01