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. 2021 Sep 20;17(9):e1009940. doi: 10.1371/journal.ppat.1009940

Fig 2. PCV2 infection results in cGAS degradation through activation of autophagy-lysosome pathway.

Fig 2

(A) PCV2 infection induces porcine cGAS reduction. PK-15 cells were infected with PCV2 (MOI = 5) or mock for the indicated time, and then the protein and mRNA levels of porcine cGAS were determined by western blotting (upper panel) and RT-PCR (lower panel). (B) Ultraviolet-inactivated PCV2 induces porcine cGAS reduction. PK-15 cells were inoculated with UV-PCV2 (MOI = 5) or mock for the indicated time, and then the levels of porcine cGAS and PCV2 capsid were determined by western blotting. (C) Chloroquine (CQ) can effectively prevent the reduction of cGAS protein in PCV2-infected cells. PK-15 cells were pretreated with 10 μM MG132 (proteasome inhibitor) or 20 μM CQ (autophagy inhibitor) for 8 h, then infected with PCV2 (MOI = 5) for indicated times to detect cGAS levels. ** P < 0.01 (compared with infection at 0 h), # P < 0.05, ## P < 0.01 (compared with DMSO treatment in indicated same time). (D, E) knockdown of Atg5 or autophagic-sequestration inhibitor 3-MA treatment alleviates PCV2-induced cGAS degradation. PK-15 cells were transfected with Atg5 specific siRNA (siAtg5) or siRNA negative control (siN.C.) for 24 h, or treated with 3-MA (5 mM) for 8 h before PCV2 infection, and then the levels of porcine cGAS were determined at the indicated times by western blotting. * P < 0.05, ** P < 0.01 (compared with infection at 0 h), # P < 0.05, ## P < 0.01 (compared with siRNA negative control or DMSO treatment in indicated same time) (F, G) cGAS proteins are co-localized with LC3 in PCV2-infected cells. PK-15 cells transfected with GFP-LC3 were infected with PCV2 in the presence of DMSO, CQ or 3-MA along, followed by confocal observation (F, Left). The co-localization signals of targeted proteins were analyzed as intensity profiles of indicated proteins along the plotted lines by Image J line scan analysis (F, Right). The number of yellow puncta was counted and compared (G). Scale bar, 10 μm. ** P < 0.01 (compared with DMSO treatment). (H) PK-15 cells were pretreated with different doses (10, 20, 40 μM) of CQ for 8 h before UV-PCV2 inoculation, and then the levels of porcine cGAS, PCV2 Cap and p62 were determined at 48 hpi.