Fig. 2: Reduced DRP1 levels and mtDNA release in presence of CoV-2 PLpro. | Cell Death & Disease

Fig. 2: Reduced DRP1 levels and mtDNA release in presence of CoV-2 PLpro.

From: ISGylation of DRP1 closely balances other post-translational modifications to mediate mitochondrial fission

Fig. 2

A Cells transfected with the indicated GFP-tagged constructs and treated with MitoTracker Red FM were imaged under live-cell conditions. Scale bar, 5μm. B Box plot showing quantification of mitochondrial length for the experiment described in A. ~90 cells from 3 independent experiments were analysed. The central line and the plus (+) symbol in each box show the median and mean value, respectively. ***p ≤ 0.001 using unpaired 2-tailed Student’s t-test. C A549 cells transiently transfected with the indicated GFP-tagged constructs were immunoblotted against the indicated antibodies. D Graphs plot changes in expression of proteins analysed in panel C. Data represent the mean ± SEM of three independent experiments. ns, not significant (p>0.1), *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 using unpaired 2-tailed Student’s t-test. E Cells were transfected with indicated GFP-tagged constructs and immunostained with antibodies against TOMM20 and TFAM. Z-stacks (0.15μm slices) were taken. Representative 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, 5μm. F Graph represents data from ∼175 cells from 3 independent experiments. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 using unpaired 2-tailed Student’s t-test. Error bars,±SEM. G A549 cells transfected similarly as in panel E and were immunoblotted against TOMM20 and TFAM antibodies, β-ACTIN was used as loading control. Graphs plot show changes in expression of proteins analysed. Data represent the mean ± SEM of 3 independent experiments. ns, not significant (p > 0.6), *p ≤ 0.05 using unpaired 2-tailed Student’s t-test. H Cytosolic and membrane fractions obtained from digitonin permeabilised A549 cells were immunoblotted with TFAM antibody; inputs indicate total protein in cell lysates. HSP90 and RTN4 served as controls for cytosolic and membrane fractions, respectively. I Graph with data from panel H shows results of 3 independent experiments. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 using unpaired 2-tailed Student’s t-test. Error bars, ±SEM. J DNA isolated from whole cell and cytosolic extracts was subjected to SYBR Green-based qPCR to quantify nuclear (GAPDH) and mitochondrial (COXII) DNA using specific primers. Plots show abundance of total (left) and cytosolic (right) cellular mtDNA. ns, not significant (p = 0.7), *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 using unpaired 2-tailed Student’s t-test. Error bars, ±SEM.

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