Figure 6.

VacA leads to early and transient accumulation of POLG and TFAM. AGS cells incubated with acid-activated VacA(wt) and VacA-(∆6-27)³⁵. (A) POLG and (D) TFAM visualized by confocal immunofluorescence (red; left panels). The right panels show merge with mitochondrial immunostaining staining (TOM22, green). Nuclei (Hoechst, blue). Scale bar: 10 µm. Quantification of POLG (B) and TFAM (E) immunofluorescence intensity (n = 30 cells/condition (and n = 50 untreated cells) from three independent experiments, mean ± SD, Welch’s test, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, versus untreated AGS). Untreated: AGS cells; Mock: AGS cells incubated with the VacA activation buffer as control. WB of (C) POLG and (F) TFAM. The 29 kDa and 25 kDa isoforms of TFAM, as well as a higher molecular weight band ( > , at ≈ 31-32 kDa) are present in all samples at 2 h, whereas at the other time points the 25 kDa isoform and the larger band are prevalent. Samples were derived from the same experiment and the gels and blots were processed in parallel (one gel for 2 h and 6 h, one gel for 24 h and 48 h). Each blot was then sliced in horizontal sections to allow multiple immunolabelings with the same membrane. Sections were alternatively labeled with anti-POLG and anti-TFAM (other labelings performed with the remaining portions of the membrane are not shown). The corresponding uncropped gels/blots are shown in Supplementary Figure S7D. SYPRO Ruby blot staining used as loading control (total protein staining is used to overcome single housekeeping protein variations) is shown in Supplementary Figure S7D (entire gel staining).