Fig. 5: SQSTM1/p62-mediated autophagy is responsible for PCV2 ORF1 degradation. | Communications Biology

Fig. 5: SQSTM1/p62-mediated autophagy is responsible for PCV2 ORF1 degradation.

From: Retinol binding protein 4 restricts PCV2 replication via selective autophagy degradation of viral ORF1 protein

Fig. 5

a Immunoblotting analysis of the indicated proteins in immunoprecipitated (IP) samples and whole-cell lysates of 3D4/21 cells transfected with PCV2 ORF1 (Myc-tagged) together with pEGFP-C1 empty vector, GFP-SQSTM1/p62, GFP-NBR1, GFP-NDP52, or GFP-OPTN for 24 h. b Immunoblotting analysis of endogenous SQSTM1 and PCV2 ORF1 in immunoprecipitated samples or whole-cell lysates of 3D4/21 cells transfected with empty vector (EV) or the PCV2 ORF1 expression plasmid. Anti-SQSTM1 immunoprecipitates or levels of the indicated proteins in whole-cell lysates were analyzed by immunoblotting with anti-Myc antibody or anti-SQSTM1 antibody. c Colocalization of PCV2 ORF1 (red) and SQSTM1, NBR1, NDP52, or OPTN (green) in 3D4/21 cells transfected with PCV2 ORF1 together with pEGFP-C1 empty vector, GFP-SQSTM1, GFP-NBR1, GFP-NDP52, or GFP-OPTN for 24 h. Scale bar, 10 μm. d Immunoprecipitation analysis of the association of PCV2 ORF1 and LC3 in 3D4/21 cells (left) or HeLa cells (right) transfected with PCV2 ORF1 and GFP-LC3 plasmids for 24 h. e Colocalization of PCV2 ORF1 (red), SQSTM1 (blue), and LC3 (green) in 3D4/21 (upper) and HeLa cells (lower). Cells were transfected with PCV2 ORF1 and GFP-LC3 for 12 h following treatment with CQ (20 μM) for another 12 h before confocal microscopy. Endogenous SQSTM1 was labeled by anti-SQSTM1 antibody. f Immunoprecipitation analysis of association of PCV2 ORF1 with LC3 in HeLa cells transfected with siRNA oligos specifically targeting SQSTM1/p62 (SQSTM1 siRNA), NBR1 (NBR1 siRNA), NDP52 (NDP52 siRNA), OPTN (OPTN siRNA), or control siRNA oligos (Control siRNA) (100 nM). Twenty-four hours after transfection, cells were further transfected with PCV2 ORF1 and GFP-LC3 plasmids for another 24 h before analysis. g Colocalization of PCV2 ORF1 (red) and LC3 (green) in HeLa cells transfected with siRNA targeting SQSTM1/p62 or control siRNA oligos (100 nM). Twenty-four hours after transfection, cells were further transfected with PCV2 ORF1 and GFP-LC3 plasmids for another 12 h followed by treated with CQ (20 μM) for another 12 h before analysis. Pearson’s correlation coefficient analysis was based on multiple sight fields in each group (n = 6 fields). h Immunoblotting analysis of PCV2 ORF1 protein and SQSTM1/p62 expression in whole-cell lysates of HeLa cells transfected with siRNA targeting SQSTM1/p62 or control siRNA oligos (100 nM). Twenty-four hours after transfection, cells were further transfected with PCV2 ORF1 for another 24 h following treatment with CHX (50 μM) for the indicated times. Densitometric quantitation of PCV2 ORF1 was normalized relative to the levels at 0 h conditions (h, right). Data are representative of three (a–e, g) or two (f, h) independent experiments or are pooled from three independent experiments (g, right). **p < 0.01, (Student’s t test).

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