Fig. 3: mtDNA release in presence of CoV2 PLpro phenocopy DRP1 and HERC5 depletion.

A Cells treated with mock or DRP1 siRNAs were immunostained with anti-TOMM20 antibody and imaged. Scale bar, 10μm. Knockdown efficiency was confirmed by immunoblotting against DRP1, cell lysates were immunoblotted against TOMM20 and TFAM, β-TUBULIN was used as loading control. Graph quantifies expression of indicated proteins. Data represent the mean ± SEM of 3 independent experiments. ns, not significant (p > 0.2) using unpaired 2-tailed Student’s t-test. Box plot with data from image panel; the central line and the plus (+) symbol in each box show the median and mean value, respectively. ~75 cells from 3 independent experiments were analysed. ***p ≤ 0.001 using unpaired 2-tailed Student’s t-test. B Representative images of cells similarly treated as in panel A were immunostained against TOMM20 and TFAM. Z-stacks (0.15μm slices) were taken. Images show 3-D projections. Enlarged views of the areas within the white boxes shown (insets). White arrowheads mark TFAM puncta outside TOMM20 boundary. Scale bar, 10μm. Graph represents data from ∼100 cells from 3 independent experiments. ***p ≤ 0.001, using unpaired 2-tailed Student’s t-test. Error bars, ±SEM. C Cells similarly treated as in panel A were stained against LIVE ORANGE mito and PicoGreen to visualise cristae and dsDNA, respectively, and imaged in the slice 3D-SIM live mode. Enlarged views of the areas within the white boxes shown (insets). White arrowheads mark PicoGreen puncta outside LIVE ORANGE mito boundary. Scale bar, 10 μm. D Cytosolic and membrane fractions obtained from digitonin-permeabilised A549 cells were immunoblotted with indicated antibodies; inputs indicate total protein in cell lysates. Note increased TFAM protein levels in cytosolic fraction and decrease in membrane fraction upon DRP1 depletion, more prominent in the blot with darker exposure. HSP90 and RTN4 served as controls for cytosolic and membrane fractions, respectively. DRP1 levels indicate knockdown efficiency. E DNA isolated from whole cell and cytosolic extracts was analysed by qPCR. Nuclear (GAPDH) and mitochondrial (COXII) DNA was quantified using specific primers. Plots show abundance of total (left) and cytosolic (right) cellular mtDNA. ns, not significant (p = 0.07), **p ≤ 0.01 using unpaired 2-tailed Student’s t-test. Error bars, ±SEM. F Mock or DRP1 siRNAs-treated cells were immunostained for ISG15. DRP1 and β-TUBULIN confirm knockdown efficiency and equal sample loading. Graph depicts changes in ISG15 levels. Data represents 3 independent experiments. ***p ≤ 0.001 using unpaired 2-tailed Student’s t-test. Error bars, ±SEM. G Representative images of cells were treated with indicated siRNAs and immunostained with antibodies against TOMM20 and TFAM. Z-stacks (0.15 μm slices) were taken. Images show 3-D projections. Insets shown. White arrowheads mark TFAM puncta outside TOMM20 boundary. Scale bar, 10μm. Graph represents data from ∼75 cells from 3 independent experiments. ***p ≤ 0.001, using unpaired 2-tailed Student’s t-test. Error bars, ±SEM. Imunoblot of lysates post-imaging confirm HERC5 depletion, VINCULIN was used as loading control. H Lysates similarly generated as in panel G were immunostained against DRP1, ISG15, HERC5 and VINCULIN. Graphs show the changes in the expression of DRP1 and ISG15. Data represents 3 independent experiments. ***p ≤ 0.001 using unpaired 2-tailed Student’s t-test. Error bars, ±SEM. I Cytosolic and membrane fractions obtained from digitonin permeabilised A549 cells were immunoblotted with TFAM antibody; inputs indicate total protein in cell lysates. Note higher levels of TFAM present in cytosolic fraction of HERC5-depleted samples. Blots representative of 3 independent experiments. HSP90 and RTN4 served as controls for cytosolic and membrane fractions, respectively. HERC5 levels confirm knockdown efficiency. J DNA isolated from whole cell and cytosolic extracts was analysed by qPCR. Nuclear (GAPDH) and mitochondrial (COXII) DNA was quantified using specific primers. Plots show the abundance of total (top) and cytosolic (bottom) cellular mtDNA. ns, not significant (p = 0.06), **p ≤ 0.01, using unpaired 2-tailed Student’s t-test. Error bars, ±SEM.