Extended Data Fig. 1: TFAM-bound nucleoids are present outside of mitochondria in TFAM knock-down and HSV-1 UL12.5-expressing cells.
a) Airyscan imaging of mtDNA FISH using a D-loop probe, followed by immunofluorescence against cGAS, HSP60, and TFAM, in IMR-90 cells transfected TFAM siRNA. Scale bars = 10 µm for larger images, 2 µm for insets. b) Quantification of extramitochondrial nucleoids positive for TFAM. N = 10 cells for both conditions, and data were quantified from one representative experiment of three. siCTRL was compared to siTFAM (p = 0.0017). c) Airyscan imaging of mtDNA FISH (D-loop probe), followed by immunofluorescence against TOM20 in U2OS cells that were transfected with either HSV-1 UL12.5 or GFP (as a negative control). Scale bars = 10 µm, ROI scale bars = 1 µm. d) Quantification of nucleoids present outside of mitochondria. GFP was compared to UL12.5 (p = 0.0005). N = 9 cells for GFP, N = 13 cells for UL12.5, and data were quantified from one experiment of three. e) Airyscan imaging of TFAM and HSP60 immunofluorescence in U2OS cells transfected with cGAS-mCherry along with either pcDNA3.1 or HSV-1 UL12.5. For the cGAS-mCherry channel, image display settings were not held constant between pcDNA3.1 and UL12.5, to allow for visualization of cGAS-mCherry structures and accounting for differences in the level of exogenous expression. Similarly, image display settings of TFAM are not constant between pcDNA3.1 and UL12.5, to account for diminished TFAM levels caused by UL12.5 expression (also causing neighboring untransfected cells to appear saturated in the bottom image shown)7. Scale bars = 20 µm, inset scale bars = 2 µm. f) Quantification of extramitochondrial nucleoids, using TFAM as a marker for mtDNA and performed similarly to quantification in D. pcDNA3.1 was compared to UL12.5 (p = 0.0068). N = 10 cells for both conditions, and data were quantified from one representative experiment of three. All differences were compared using unpaired, two-tailed student’s t test. For all plots, lines represent mean. Source numerical data are available in source data.
