Extended Data Fig. 4: Enlarged nucleoids are not released through VDAC pores.
a) qRT-PCR of primary Tfam+/−MEFs transfected with control, VDAC1, or VDAC3 siRNAs, normalized to β actin. Dots represent biological replicates of independent experiments (N = 3). b) Western blotting of VDAC1 and β actin (loading control) in primary MEFs transfected with control or VDAC1 siRNAs. c) Quantification of B. Dots represent biological replicates (N = 3). d) qRT-PCR of Vdac3 in primary Tfam+/−MEFs transfected with control or VDAC3 siRNAs, normalized to β actin. qPCR data is shown due to the fact that attempts to blot for VDAC3 failed. Dots represent biological replicates of independent experiments (N = 3). e) U2OS cells expressing HSV-1 UL12.5 and cGAS-Halo were treated with either DMSO or VBIT4 (10 µM) for 24 hrs and imaged for TFAM and HSP60 immunofluorescence using resonance scanning confocal microscopy. The number of extramitochondrial cGAS+ TFAM+ puncta was scored, and DMSO was compared to VBIT4 by unpaired, two-tailed t test (p = 0.6405). N = 50 cells for DMSO and N = 49 cells for VBIT4, and data were quantified from three independent experiments. f) Western blotting against PARP in cells treated with the indicated concentration (100 nM or 500 nM) of the apoptosis inducer ABT-737 along with either DMSO or VBIT4 (10 µM). The reduction of cleaved PARP indicates that the induction of apoptosis is reduced in cells treated with VBIT4, as previously published63, demonstrating that the VBIT4 used in panel E is active. β actin was probed as a loading control. Results were reproduced across three independent experiments. For all plots, data are reported as mean ± SEM. Source numerical data and unprocessed blots are available in source data.
