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Figure 4: Involvement of PI3K signaling in the reduction of the expression of TLR ligands and elements of TLR signaling pathway in U937 cells to Mtb H37Rv infection in the A549 cell coculture model. In the presence or absence of PI3K inhibitor LY294002, the coculture model of A549/U937 cells was infected with H37Rv mycobacteria from the upper chamber (A549 cells, AI), lower chamber (U937 cells, UI), or both chambers (A549 and U937 cells, CI) at a MOI of 3 for 18 h before the culture medium and U937 cells were harvested for analysis. (a) Representative blots of immunoblotting assay for the indicated components of TLR signaling cascade showed a reversed TLR signaling activity in U937 cells of the coinfection model in the presence of LY294002, in comparison with the absence of an inhibitor. (b) The fold of changes of proteins of interest in (a) semiquantitatively determined by densitometric assay using ImageJ software Fiji from three independent experiments. The ability of A549 cell-mediated reduction of TLR signaling activity in U937 cells was reversed by the addition of LY294002. (c) Concentrations of TNF-α, IL-10, and IL-6 in culture media determined by ELISA; the A549 cell-mediated reduction of cytokines in H37Rv-infected U937 cells was reversed in the presence of PI3K inhibitor. Error bars represent the standard deviation (SD) from three independent experiments. Compared to noninfection (NI) control, and ; compared to the absence of LY294002, and . NI: noninfected control; AI: infection was performed on A549 cell alone; UI: infection was performed on U937 alone; CI: infection was performed on both A549 cells and U937 cells.