Figure 5.

Loss of G2A does not induce enhanced phagosome maturation or intracellular Ca²⁺ release in lysophosphatidylcholine (LPC)-treated macrophages during H37Ra infection. (A) Wild-type (WT) and G2A knockout (KO) bone marrow-derived macrophages (BMDMs) were infected with H37Ra [multiplicity of infection (MOI) of 5] and treated with LPC for 30 min. Cells were then loaded with calcium sensing Fluo-4/AM and stained with diamidino-2-phenylindole to identify nuclei. All images were observed by confocal microscopy. The bar graph represents the ratio of the Fluo-4/AM mean fluorescence intensity (MFI), which was normalized to the MFI obtained for uninfected cells. (B) Raw264.7 cells were pretreated with BAPTA/AM (30 µM) for 30 min and stimulated with LPC during fluorescein isothiocyanate (FITC)-labeled H37Ra infection (MOI of 5). After infection, cells were fixed and stained with early endosomal antigen 1 (EEA1) and lysosomal-associated membrane protein 1 (LAMP-1). The bar graphs represent the ratio of Mycobacterium tuberculosis (Mtb) colocalization with each marker. (C) WT and G2A KO BMDMs were infected with FITC-labeled H37Ra with or without LPC treatment for 3 h. After infection, cells were fixed and stained with EEA1 and LAMP-1. All images were viewed under a confocal microscope. The bar graphs represent the ratio of Mtb colocalization with each marker (**p < 0.01 and ***p < 0.001).