Fig. 3: VZV gE modulates mitochondria dynamics and regulated PINK1/Parkin-dependent mitophagy.

A HeLa-Parkin cells were transiently transfected with EV or VZV gE (green) along with mt-dsRED encoding plasmid (red) for 24 h and then treated with CCCP for 2 h. Quantification of the mitochondrial area is shown in the graph. **p < 0.01, ***p < 0.001 vs. EV-transfected cells. B HeLa-Parkin cells expressing either EV or VZV gE were subjected to immunoblot analysis. Whole-cell lysates (WCL) and mitochondrial fractions (mito) were isolated and VZV gE, PINK1, Parkin, phospho-DRP1(Ser616), DRP1, MFN1, MFN2, and TOM20 expression was measured. A representative of three independent experiments is shown. β-actin was used as a protein loading control. C p62, LC3, and TOM20 protein levels were examined in VZV gE transfection in HeLa-Parkin cells by immunoblot analysis. D HEK293T cells (Ctl) or HEK293T stably expressing VZV gE (gE) were treated with DMSO (Ctl) or CCCP for 2 h (left). MRC5 cells were infected with VZV (MOI 0.001) for 48 h (right). The ubiquitination of Parkin was measured by immunoblot assay using anti-phospho-ubiquitin (Ser65), Parkin, and VZV gE-specific antibodies. E Cells were transiently transfected with the indicated amount of VZV gE plasmid for 24 h and then treated with Ctl or CCCP for 2 h. Relative tetramethylrhodamine methyl ester (TMRM) intensities were measured. **p < 0.01; ***p < 0.001 vs. EV-transfected Ctl-treated cells. #p < 0.05 vs. VZV gE-transfected Ctl-treated cells. F HeLa-Parkin cells expressing mtKeima were transfected with EV or VZV gE plasmids along with siRNA control (siCtl) or siPINK1 (siPINK1) for 24 h. After CCCP treatment for 2 h, mitophagy levels were determined by confocal microscopy. *p < 0.05; **p < 0.01; ***p < 0.001 vs. EV-transfected cells. #p < 0.05 vs. gE-transfected siCtl-expressing cells. G Mitochondrial DNA (mtDNA) content of VZV gE-expressing HeLa-Parkin cells was analyzed by RT-qPCR (mean ± SD; n = 3). ***p < 0.001 vs. EV-transfected cells. H HeLa-Parkin cells were transiently transfected with EV or VZV gE plasmid; 24 h later, the cells were either stimulated with DMSO (Ctl) or 3 μM staurosporine (STS) for 2 h. Cleavage of PARP, caspase 3, 7, and 8 is shown by immunoblot analysis. β-actin was used as a protein loading control. Images are representatives of three independent experiments. I Activities of caspases 3/7 or caspase 8 in EV or VZV gE-expressing cells treated with STS was determined using caspase luminescence assays. Data represented as mean ± SD. **p < 0.01; ***p < 0.001 vs. EV-transfected cells in each group. J Flow cytometry analysis by Annexin V-FITC/PI staining showed % cells undergoing apoptosis in HeLa-Parkin, HeLa-shPINK1, and PINK1-V5-transfected HeLa-shPINK1 (HeLa-shPINK1 + PINK1) cells.