Figure 5: Mitochondrial-targeted PB1-F2 and its effect on Δψ_m.

(a) HEK293 cells were infected with A/PR8 (16 HA U ml⁻¹) for about 15 h, and the cells were stained with the cationic fluorescent dye TMRM and analyzed by flow cytometry (right panel; red trace). As a control, CCCP-treated cells (40 μM) were also analyzed (left panel). Grey histograms in both panels represent a profile of unstained cells. (b) Similar to a, except that HEK293 cells were transfected with the expression plasmid indicated inside the panel. Cells were analyzed by flow cytometry at 24 h post transfection. (c) The kinetics of Δψ_m disruption in A/PR8-infected J774A.1 macrophages. Cells infected with A/PR8 were collected at the indicated time points (0, 3, 6, 9, 15, 18 and 21 h), stained with JC-1 for 30 min, and analyzed by flow cytometry. The percentage of JC-1 reduction (y axis) is presented. The immunoblot on the right represents a profile of PB1-F2 expression at each time point as well as the loading controls β-actin and OPA-1. Five bands (a–e) of OPA-1 isoforms were detected by immunoblotting with the antibody against OPA-1, and bands a and b are a mixture of L-OPA-1 isoforms. (d) Δψ_m is dispensable for PB1-F2 translocation into mitochondria. HeLa cells transfected with either mitochondrial-targeted dihydrofolate reductase (mtDHFR) or PB1-F2 were treated with (+) or without (−) CCCP (40 μM), and their translocation into mitochondria was monitored by immunofluorescence microscopy (left images). Quantification is listed in the right score panel. In each transfection experiment, at least 100 cells were scored, and the green and red bars represent mitochondrial and cytosolic localization, respectively. All data represent the mean values±s.d. (n=3 experiments). Scale bar, 10 μm. N.D., not detected, **P<0.01 and ***P<0.001 (by unpaired t-test).