NOTCH1 signaling is essential for mycobacteria-mediated MSI expression and FM formation.

<p>(A) Mouse peritoneal macrophages were treated with specific inhibitors of NOTCH1 activation (GSI), PI3K (LY294002), mTOR (Rapamycin), NF-κB (BAY 11–7085), SHH signaling (Cyclopamine, Betulinic Acid) and WNT signaling (IWP-2, FH535) 60 min prior to 6 h infection with BCG. Immunoblotting for MSI was performed. (B and C) Peritoneal macrophages treated with specific inhibitors were infected with H37Rv expressing the red fluorescent protein tdTomato for 48 h. Representative IF images of macrophages with BODIPY-stained lipid droplets (B). Based on the IF images, MFIs were calculated (n = 100, each treatment) and plotted (C, left panel). Frequency of FMs was calculated by counting the population of cells expressing 0–5, 5–10 or >10 lipid bodies (n = 250–300) and plotted as a bar graph (C, right panel). (D) Activation analysis of NOTCH-PI3K-mTOR-NF-κB pathway by immunoblotting in peritoneal macrophages from WT or <i>tlr2</i>-KO mice (left panel) and RAW 264.7 cells transfected with <i>Notch1</i>-specific siRNAs (right panel) after 1 h of BCG infection. (E-H) Murine RAW 264.7 macrophages transfected with <i>Notch1</i>-specific siRNAs were infected with BCG for 12 h and analyzed for the expression of the indicated genes by immunoblotting (E and F) and quantitative real-time RT-PCR (G). Immunoblotting for NOTCH1 to validate <i>Notch1</i> siRNA (H). (I) IF imaging of BODIPY-stained lipid droplets in peritoneal macrophages transfected with NT or <i>Notch1</i> siRNA and infected with H37Rv expressing the red fluorescent protein tdTomato for 48 h (I, left panel). MFIs (top right panel) and frequency of FMs (bottom right panels) were calculated as mentioned in panel C. All data represents the mean ± SEM for at least 3 independent experiments, *<i>P</i> < 0.05, **<i>P</i> < 0.005, ***<i>P</i> < 0.0005 (one-way ANOVA followed by Tukey’s multiple-comparisons test) and all blots are representative of 3 independent experiments. Med, medium; WT, wild-type; KO, knockout; NT, non-targeting; MFI, mean fluorescence intensity. Bar, 5 μm; Original magnifications 63X.