Figure 3

Transient alteration of mitochondrial translocases early upon H. pylori infection. AGS cells co-cultured with H. pylori 26695 and 26695∆vacA. (A) 3D-reconstructed immunofluorescence of cells labelled with VacA (purple), TOM22 (green), and counterstained with Hoechst (nuclei, blue). (B) Quantification of VacA immunofluorescence. Extramitochondrial VacA (VacA⁺/TOM22^-, hatched) and intramitochondrial VacA (VacA⁺/TOM22⁺, black); error bars in red; no detection of VacA (“nd” for “not detected”) in non-infected cells and cells infected with the H. pylori 26695∆vacA. For infection with Hp26695, intramitochondrial VacA (in black in the histogram) was also tested at different time points with one-way ANOVA and Dunn’s multiple comparisons test, in both cases resulting in p < 0.0001 (**** in red); differences in total VacA in these conditions were not significant. (C) Percentage of cells displaying mitochondrial fragmentation (n = 60 cells/condition, from 3 independent experiments, 2-tailed Fisher’s exact test). Insets (5x magnification) illustrate fragmented (white arrowheads) and non-fragmented (yellow arrows) mitochondria. (D) Quantification of TOM22 immunofluorescence intensity/cell. (E) MtDNA content quantified by qPCR (mean ± SD). (F) 3D-reconstructed fluorescence of cells labelled with MitoTracker Deep Red (purple), TIM23 immunostaining (green), and nuclei (Hoechst, blue) and fluorescence quantification. In panels D and F, mean ± SD, n = 30 cells/conditions from 3 independent experiments, Welch’s test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Scale bar: 10 µm.