Figure 9.

Inhibition of LDHB promotes CSFV replication via mitophagy. Overexpress or knockdown LDHB has the opposite effect on CSFV growth. PK-15 (A and C) and 3D4/2 (B and C) cells were transduced with the siNC or siLDHB (left), p3xflag-LDHB or p3xflag-CMV (right) for 24 h, followed by infected with CSFV at a MOI of 0.1 or mock-infected. (A and B) At 24 hpi, cell samples were analyzed by immunoblotting with antibodies against LDHB, CSFV Npro, Flag and TUBA (loading control). The level of protein was quantified using Image-Pro Plus 6.0 software. Error bars indicate the mean (±SD) of 3 independent experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 (one-way ANOVA). (C) At 24 hpi, the CSFV genome copy numbers in PK-15 and 3D4/2 cells were assessed using a real-time RT-PCR assay as described in Materials and Methods. CSFV virus titers in the supernatant were determined as 50% tissue culture infective doses (TCID₅₀)/mL as described in Materials and Methods. Error bars indicate the mean (±SD) of 3 independent experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 (one-way ANOVA). (D) PK-15 and 3D4/2 cells were treated with CCCP, BAY or shPRKN before transfected with siLDHB, followed by infected with CSFV at a MOI of 0.1. At 24 hpi, cell samples were analyzed by immunoblotting with antibodies against CSFV Npro and TUBA (loading control). The level of protein was quantified using Image-Pro Plus 6.0 software. Error bars indicate the mean (±SD) of 3 independent experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 (one-way ANOVA). (E) PK-15 and 3D4/2 cells were treated as (D), At 24 hpi, the CSFV genome copy numbers in PK-15 and 3D4/2 cells were assessed using a real-time RT-PCR assay as described as (C)