Fig. 8

BNIP3 and CTSE transcriptomic and western blot analyses with RNAs and protein extracts from L. m.-infected and HBSS-starved BMDM as well as determination of the infection rates of L. m.-infected BMDM after BNIP3 and CTSE downregulation by RNA interference. Methods: (a, b) BMDM from BALB/c mice were infected with L. m. promastigotes for 1 h or 24 h. Uninfected control BMDM were incubated for the same amount of time in RPMI medium or starved for 1 h in HBSS. The Proteins were harvested and subjected to western blot analysis with specific antibodies against BNIP3 and CTSE. Western blots 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. Affymetrix® chips were hybridized with RNA samples from 2 independent experiments and analyzed densitometrically. c Additionally, L. m.-infected BMDM were transfected with specific siRNAs 20 h p.i. to downregulate the expression of BNIP3 and CTSE. L. m.-infected control BMDM were transfected with negative control siRNA. Infection rates were determined 48 h p.i. in 2 independent experiments. Results: (a) BNIP3 and CTSE were significantly overexpressed in L. m.-infected BMDM 24 h p.i. compared to uninfected control BMDM. b Densitometric analyses of western blot experiments confirmed this overexpression 24 h p.i. and showed that CTSE was also overexpressed in L. m.-infected BMDM 1 h p.i.. At the mRNA level, Bnip3 was overexpressed in L. m.-infected BMDM 1 and 24 h p.i. and Ctse was downregulated in L. m.-infected BMDM 24 h p.i.. c A significant increase in the infection rates was detected in L. m.-infected BMDM after downregulation of protein expression of BNIP3 or CTSE 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,*** p ≤ 0.001