Fig. 4.

Nucleolin mediates SARS-CoV-2 induction of the stress response and apoptosis. Western blot determination of NCL, apoptotic, stress response and DNA damage proteins in total cell lysate (A) and nuclear and cytoplasmic fractions (B) of Vero E6-TMPRSS cells, control and NCL-KD infected with mock and SARS-CoV-2 (MOI 0.01) for 48 h. β-actin: loading control. Rb and GAPDH were used as nuclear and cytoplasmic markers, respectively. C. Flow cytometric determination, in triplicate, and ImageJ quantification of apoptosis by staining of Vero E6-TMPRSS control and NCL-KD cells with annexin V-Alexa 488, propidium iodide (PI) and mitotracker conjugated with phycoerythrin (PE). The number of positive cells is shown (%). D. Western blot determination in triplicate and ImageJ quantification of NCL, N-protein and cytochrome C (Cyt c) in cytoplasmic and mitochondrial fractions of Vero E6-TMPRSS cells, control and NCL-KD, infected with mock and SARS-CoV-2 (as above). The ratio of Cyt c expression in cytoplasm (C) by mitochondria (M) is shown. E. Merged confocal immunofluorescence image of cytochrome c (Cyt c, green), mitochondrial marker Cox IV (red) and Hoechst (blue) in Vero E6-TMPRSS cells infected with SARS-CoV-2 (MOI 0.01) for 48 h. The student t-test was performed. *P < 0.05, **P < 0.01 and ***P < 0.001.