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. 2021 Nov 25;18(8):1801–1821. doi: 10.1080/15548627.2021.2002101

Figure 6.

Figure 6.

Inhibition of USP14 induces autophagy. (A) PK-15 cells were treated with b-AP15 (0–1 μM) for 24 h. LC3-I, LC3-II, SQSTM1, ATG5, ATG12 and BECN1 were assessed by immunoblot analysis. (B) LC3-I, LC3-II, SQSTM1, ATG5, and BECN1 were assessed by immunoblot analysis in sgControl and sgUSP14 PK-15 cells. (C) PK-15 cells were transfected with plasmid encoding GFP-LC3 and treated with DMSO or b-AP15 (1 μM) for 24 h. The fluorescence of GFP-LC3 was detected by fluorescent microscopy. Scale bar: 10 μm. (D) sgControl and sgUSP14 PK-15 cells were transfected with plasmid encoding GFP-LC3 for 24 h. The fluorescence of GFP-LC3 was detected by fluorescent microscopy. Scale bar: 10 μm. (E) Quantification of GFP-LC3 puncta per cell from C and D using ImageJ software. (F) PK-15 cells were treated with DMSO or b-AP15 (1 μM) for 24 h. The autophagosome-like vesicles were detected by transmission electron microscope. Scale bar: 500 nm. (G) Quantification of autophagosome-like vesicles from F using ImageJ software. (H) PK-15 cells were transfected with plasmid encoding GFP-RFP-LC3 and treated with DMSO or b-AP15 (1 μM) for 24 h. The fluorescence of GFP and RFP was detected by fluorescent microscopy. Scale bar: 10 μm. (I) Quantification of autophagosomes and autolysosomes from H using ImageJ software. (J) sgControl and sgUSP14 PK-15 cells were treated with bafilomycin A1 (10 μM) for 24 h. LC3-I, LC3-II and SQSTM1 were assessed by immunoblot analysis. (K) ATG5 in sgControl and sgATG5 PK-15 cells was assessed by immunoblot analysis. (L) BECN1 in sgControl and sgBECN1 PK-15 cells was assessed by immunoblot analysis. (M and N) sgControl, sgATG5 (M) and sgBECN1 (N) PK-15 cells were infected with PRV-QXX (MOI = 0.1) and treated with b-AP15 (1 μM) as indicated for 24 h. PRV VP16 was assessed by immunoblot analysis. (O) sgControl, sgATG5 and sgBECN1 PK-15 cells were infected with PRV-QXX (MOI = 0.1) and treated with DMSO or b-AP15 (1 μM) as indicated for 24 h. Viral titers were assessed by the TCID50 assay. Data were shown as mean ± SD based on three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 determined by two-tailed Student’s t-test. ns, no significance.