FIG 3.

TMEM41B knockdown inhibits PRV infection. (A) The level of TMEM41B mRNA expression was assessed in scramble, shTMEM41B-1, and shTMEM41B-2 PK-15 cells by qRT-PCR analysis. ***, P < 0.001. (B) TMEM41B and nectin-1 were assessed in scramble, shTMEM41B-1, and shTMEM41B-2 PK-15 cells by immunoblotting analysis. (C) Membrane distribution of nectin-1 in scramble, shTMEM41B-1, and shTMEM41B-2 PK-15 cells was analyzed by a nonpermeabilizing immunofluorescence analysis. Scale bar = 10 μm. (D) Quantification of the relative intensity of nectin-1 on the PM from panel C by ImageJ (n = 15). The intensity of nectin-1 on the PM from scramble PK-15 cells was normalized to 1. (E) Cell viability of scramble, shTMEM41B-1, and shTMEM41B-2 PK-15 cells was assessed by a CCK-8 assay for 0 to 60 h. (F) Morphology of scramble, shTMEM41B-1, and shTMEM41B-2 PK-15 cells. Scale bar = 10 μm. (G) Scramble, shTMEM41B-1, and shTMEM41B-2 PK-15 cells were infected with PRV-GFP (MOI = 1) for 20 h. Viral replication was analyzed by fluorescence microscopy of GFP expression. Scale bar = 100 μm. (H) GFP-positive cells from E were analyzed by flow cytometry. ***, P < 0.001. (I) Scramble, shTMEM41B-1, and shTMEM41B-2 PK-15 cells were infected with PRV-QXX (MOI = 1) for 24 h. TMEM41B and PRV gB were assessed by immunoblotting analysis. (J) Scramble, shTMEM41B-1, and shTMEM41B-2 PK-15 cells were infected with PRV-QXX (MOI = 1) for 24 h. The level of PRV gB and TK mRNA expression was assessed by qRT-PCR analysis. ***, P < 0.001. (K) Scramble, shTMEM41B-1, and shTMEM41B-2 PK-15 cells were infected with PRV-QXX (MOI = 0.1 and 1) for 24 h. Viral titers were assessed by a TCID₅₀ assay. ***, P < 0.001.