Figure 10.

NF-κB overexpression significantly ameliorates cell death and mitochondrial pathology induced by MIF depletion in AGS cells. A, flow cytometric analysis to follow mitochondrial transmembrane potential (ΔΨm) in empty vector and siMIF and siMIF + pNF–κB p65 treatment in AGS cells. pNF–κB p65 is the NF-κB overexpression plasmid; the red signal in the scatter plot indicates JC-1 aggregates fluorescing at 590 nm, and green indicates JC-1 monomers (corresponding to depolarized mitochondria) fluorescing at 530 nm. Percentage values represent the number of cells emitting red or green signals, corresponding to cells with polarized or depolarized mitochondria. 10,000 events/experimental set were screened, and a representative flow cytometry scatter plot of the gated cell population is presented. B, high-resolution confocal micrographs demonstrate mitochondrial fragmentation in empty vector and siMIF- and siMIF + pNF-κB p65–treated AGS cells. Scale bar, 10 μm. 80–100 cells were randomly screened, and a single field was randomly selected for demonstration. Enlarged images of the ROI were prepared by digital zooming of the selected region for clear visualization of mitochondrial filaments. Quantification of the mitochondrial length distribution of control and MIF-silenced cell by LAS-X software is presented below each set of micrographs. 80–100 cells were screened for the analysis. Scatter plots in the lower right quadrant of each panel represent mitochondrial length distribution. Each color represents a specific filament length. C, flow cytometric analysis of apoptosis in AGS cells treated with empty vector or siMIF or siMIF + pNF-κB p65 with the help of FITC–annexin V/PI staining. 10,000 events/experimental set were screened, and a representative flow cytometry scatter plot of the gated cell population is presented. Quadrants Q2 and Q4 correspond to late and early apoptosis, respectively, and cumulatively represent annexin V binding to cells undergoing apoptosis. Percentages of cells are presented in each respective quadrant. The data presented are representative of three independent experiments. All experiments were done in triplicate. The details of each method are given under “Experimental procedures.” PE, phycoerythrin. ns = nonsignificant; *, p < 0.05 and **, p < 0.01 versus control; #, p < 0.05 and ##, p < 0.01 versus siMIF treatment calculated by ANOVA followed by Bonferroni's post hoc test.