Figure 7. Inhibition of autophagy with specific siRNA targeting endogenous LC3 enhances TGEV replication.

(a) ST cells were transfected with siRNA duplexes against porcine LC3 or nontargeting control siRNA, as described in Material and Methods. Knockdown efficiency of LC3 siRNA duplexes was indicated by relative LC3 mRNA to β-actin mRNA when siRNA duplexes at the concentration of 100 nM. (b) Detergent lysate from equivalent numbers of ST cells treated with LC3-specific or nontargeting siRNA were subjected to reducing SDS-PAGE and immunoblotting with antibodies to LC3 or β-actin (loading control). (c) ST cells were transfected with LC3-specific siRNA duplexes 3# or control siRNA at the concentration of 100 nM for 24 h followed by infection with TGEV. At 24 h post infection, cell monolayer was fixed and examined the TGEV infection by IFA as described in Fig. 6b legend. The percentage of TGEV-infected cells per view from three independent experiments is expressed as mean ± SD. (d) Determination of TGEV replication in LC3-specific siRNA-treated cells. The extracellular virus yields were determined at 24 hpi and expressed as TCID₅₀ per 0.1 ml. Representative data from three independent experiments are shown as the mean ± SD. *p < 0.05. The p value is calculated using Student’s t-test. Gels were run under the same experimental conditions. For better clarity and conciseness of the presentation, cropped blots are shown. The raw uncropped images can be found in the Supplementary Fig. S5.