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

Figure 7.

Figure 7.

Inhibition of USP14 induces ER stress. (A) PK-15 cells were treated with b-AP15 (0–1 μM) for 24 h. HSPA5, ATF6, p-EIF2AK3, EIF2AK3, p-EIF2A, EIF2A, ATF4, XBP1 and FOXO1 levels were assessed by immunoblot analysis. (B) XBP1, FOXO1, p-EIF2AK3, EIF2AK3, p-EIF2A and EIF2A levels were assessed by immunoblot analysis in sgControl and sgUSP14 PK-15 cells. (C) PK-15 cells were treated with b-AP15 (1 μM) and GSK2606414 (10 μM) as indicated for 24 h. HSPA5, ATF6, p-EIF2AK, EIF2AK3, p-EIF2A, EIF2A, XBP1, LC3-I, LC3-II, SQSTM1 and ATG5 were assessed by immunoblot analysis. (D) sgControl and sgUSP14 PK-15 cells were treated with GSK2606414 (10 μM) as indicated for 24 h. HSPA5, ATF6, p-EIF2AK3, EIF2AK3, p-EIF2A, EIF2A, XBP1, LC3-I, LC3-II, SQSTM1 and ATG5 and USP14 were assessed by immunoblot analysis. (E) sgControl and sgUSP14 PK-15 cells were mock infected or infected with PRV-QXX (MOI = 0.1) for 24 h. USP14, VP16, XBP1, FOXO1, p-EIF2AK3, EIF2AK3, p-EIF2A, EIF2A, SQSTM1, LC3-I and LC3-II were assessed by immunoblot analysis. (F) PK-15 cells were infected with PRV-QXX (MOI = 0.1) and treated with b-AP15 (1 μM) and GSK2606414 (10 μM) as indicated for 24 h. PRV VP16 was assessed by immunoblot analysis. (G) PK-15 cells were transfected with plasmid encoding FLAG-VP16 and treated with b-AP15 (1 μM) and GSK2606414 (10 μM) as indicated for 24 h. FLAG-VP16 was assessed by immunoblot analysis. (H) sgControl and sgUSP14 PK-15 cells were infected with PRV-QXX (MOI = 0.1) and treated with GSK2606414 (10 μM) for 24 h. PRV VP16 was assessed by immunoblot analysis. (I) PK-15 cells were infected with PRV-QXX (MOI = 0.1 and 1) and treated with DMSO, b-AP15 (1 μM), GSK2606414 (10 μM) and b-AP15 (1 μM) + GSK2606414 (10 μM) for 24 h. Viral titers were assessed by the TCID50 assay. (J) PK-15 cells were transfected with siControl, siEIF2A-1, siEIF2A-2 and siEIF2A-3 for 48 h. EIF2A was assessed by immunoblot analysis. (K) PK-15 cells were transfected with siControl or siEIF2A-1 and treated with b-AP15 (1 μM) as indicated for 48 h. p-EIF2A, EIF2A, LC3-I, LC3-II, SQSTM1 and ATG5 were assessed by immunoblot analysis. (L) PK-15 cells were transfected with siControl or siEIF2A-1 for 24 h. Then, cells were infected with PRV-QXX (MOI = 0.1) and treated with b-AP15 (1 μM) as indicated for 24 h. p-EIF2A, EIF2A, VP16, LC3-I, LC3-II, SQSTM1, and ATG5 were assessed by immunoblot analysis. Data were shown as mean ± SD based on three independent experiments. ** P < 0.01, *** P < 0.001 determined by two-tailed Student’s t-test. ns, no significance.