Skip to main content
. 2021 Jun 21;12:3818. doi: 10.1038/s41467-021-24007-w

Fig. 2. SARS-CoV-2 limits autophagy signaling and blocks autophagic flux.

Fig. 2

a Protein levels and phosphorylation status of selected autophagy-relevant proteins in SARS-CoV-2-infected VeroFM cells at 8 h, 24 h, or 48 h post infection (h p.i.) were analyzed by Western blotting. For analysis of ATG14 oligomers (virtual blot, bottom panel, left) proteins were cross-linked 2 h prior to cell harvest and analyzed by Wes (ProteinSimple) capillary electrophoresis 8-48 h p.i. P-values were determined by one-way ANOVA, Bonferroni post hoc test. b Fluorescence microscopy of transfected and SARS-CoV-2-infected VeroFM cells expressing pH-sensitive tandem fluorescent-tagged LC3B-mRFP/EGFP showed that low pH autophagolysosomes (AL, red) were reduced compared to autophagosomes (AP, green + red = yellow) in virus-infected cells. Microscopic read-out was done by a scientist blind to the experimental conditions. For mock (n = 44 cells) and SARS-CoV-2-infected (n = 46 cells) VeroFM cells were analyzed. P-values were determined by two-way ANOVA, Tukey´s post hoc test, mean with SEM. Scale bar = 10 µM. c Accumulation of autophagy marker P62 in SARS-CoV-2-infected VeroFM cells. d Elevated LC3B-II levels in SARS-CoV-2-infected and bafilomycin A1 (BafA1)-pretreated VeroFM cells indicate virus-induced autophagic flux inhibition at 8 and 24 h p.i. In all panels, error bars denote SEM derived from n = 3 biologically independent samples derived from one experiment. P-values were determined by two-way ANOVA, Sidak post hoc test. p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***), p ≤ 0.0001 (****), p > 0.05 (not significant, ns). Abbreviations: AKT1, RAC-alpha serine/threonine-protein kinase; AMPK, AMP-activated protein kinase; ATG14, autophagy-related 14; ATG16L1, autophagy-related 16 like 1; BafA1, bafilomycin A1; BECN1, Beclin-1 protein; CDK2, cyclin-dependent kinase 2; EGFP, enhanced green fluorescent protein; LC3B, microtubule-associated protein 1 A/1B light chain 3B; LXRXX(pS/pT), phospho-AMPK substrate motif; mRFP, monomeric red fluorescent protein; mTORC1/2, mechanistic target of rapamycin complex 1/2; PRAS40, proline-rich AKT1 substrate 1; Rheb, Ras homolog enriched in brain; SKP2, S-phase kinase-associated protein 2; TSC1/2, tuberous sclerosis 1/2; ULK1, Unc-51-like kinase 1; VPS34, phosphatidylinositol 3-kinase catalytic subunit type 3.