Figure 7. Induction of autophagy with rapamycin enhances CSFV replication. (A and D) PK-15 (A) and 3D4/2 (D) cells were pretreated with rapamycin (100 nM) or DMSO (Control) for 1 h, followed by CSFV adsorption for 1 h at an MOI of 0.5. The cells were further cultured in fresh medium in the absence or presence of rapamycin (100 nM). At 24 and 48 hpi, cell samples were analyzed by immunoblotting with antibodies against LC3B, CSFV-E2, and ACTB (loading control). The relative levels of the targeted proteins were estimated by densitometric scanning, and the ratios were calculated relative to ACTB. The data represent the mean ± SD of 3 independent experiments. Two-way ANOVA; *P < 0.05; **P < 0.01. (B and E) PK-15 (B) and 3D4/2 (E) cells were pretreated and infected as described in (A and D). At 24 and 48 hpi, both the extracellular and intracellular copy numbers of CSFV were detected by qRT-PCR. The data represent the mean ± SD of 3 independent experiments. Two-way ANOVA; *P < 0.05; **P < 0.01; ***P < 0.001. (C and F) PK-15 (C) and 3D4/2 (F) cells were pretreated and infected as described in (A and D). At 24 and 48 hpi, both the extracellular and intracellular virus titers were measured by endpoint dilution titrations by using an immunofluorescence assay as described in Materials and Methods. Results are expressed in units of TCID₅₀/ml. The data represent the mean ± SD of 3 independent experiments. Two-way ANOVA; *P < 0.05; **P < 0.01; ***P < 0.001.