Extended Data Fig. 4: Characterization of the impacts of vBcl-2 on mitochondrial morphology.
a, Confocal micrographs of mitochondria in iSLK-BAC16.vBcl-2 WT/KO cells induced by Dox/NaB for indicated time and stained with MitoTracker (red) and DAPI (blue). b, Quantification of mitochondrial morphology (tubulated, intermediate, and fragmented) in a (n = 3, with 100 cells/experiment). c-e, Analysis of mitochondrial numbers per cell (c), mean area (d) and perimeter (e) in cells in a (n = 94 cells/group, 3 independent experiments). f,g, Confocal micrographs of mitochondria (red) in cells transduced with indicated shRNAs plus NM23-H2 rescue plasmids post-reactivation (f) with corresponding quantification of fragmentation (g; n = 3, 300 cells/sample). h, IB of the indicated mitochondrial fission and fusion factors in mitochondrial fractions and WCL from iSLK-BAC16.vBcl-2 KO cells stably expressing HA-vBcl-2 (WT/mutants) at 60 h post-reactivation. TOM20, control for mitochondrial fraction. GAPDH, a loading control for WCLs (see Source Data for uncropped WB). i, Confocal micrographs of mitochondria (TOM20, green) in iSLK-BAC16.vBcl-2 KO cells expressing mCherry-vBcl-2 (red) and transduced with Vec, an MFN2-encoding plasmid, or with Ctrl shRNA, DRP1 shRNA, MFF shRNAs at 60 h post- reactivation. j, Percentage of cells in (i) with mitochondrial fission was quantified (n = 3, with 100 cells/experiment). k, Infectious virus titer in cells in (i) with MFF depletion was quantified by infecting SLK cells (top, n = 3) and GFP images are shown (left). Scale bars, 10 μm. Data in a,f,h,i,k are from one experiment that is representative of three independent experiments. Data in b,g,j,k represent mean ± s.d. analyzed by one-way ANOVA followed by Tukey’s post hoc test. Box-plot data in c-e are presented as median (center line), interquartile range (IQR; box, 25th to 75th percentiles), and whiskers extending from minimum to maximum values; and analyzed using Kruskal-Wallis with post hoc Dunn’s test. ***, p < 0.001; ****, p < 0.0001; ns, not significant. Exact P values are provided in Source Data Extended Data Fig. 4.
