Cholesterol Biosynthesis Modulates CSFV Replication

Figures

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  Figure 1. The alterations of intracellular cholesterol in CSFV-infected PK-15 cells. (a) CSFV one-step growth curve in PK-15 cells; n = 3; **** p < 0.0001. (b) Variation curve of intracellular cholesterol contents in CSFV-infected PK-15 cells; n = 3; *** p < 0.001; **** p < 0.0001. (c) Correlation analysis of CSFV replication and intracellular cholesterol levels. (d) The CSFV E2 protein and intracellular cholesterol levels in CSFV-infected PK-15 cells were assessed by IFA and Nile red staining. (M, mock; C, CSFV).

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  Figure 2. Cholesterol biosynthesis in CSFV-infected PK-15 cells. (a) GO and KEGG enrichment for differentially expressed genes in CSFV-infected PK-15 cells. (b,c) The expression of gene involved in cholesterol biosynthesis was detected in CSFV-infected PK-15 cells; ** p < 0.01; *** p < 0.001; **** p < 0.0001. (M, mock; C, CSFV; Rel. expr. lvl, relative expression level).

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  Figure 3. PCSK9 expression and exogenous cholesterol uptake during CSFV infection. (a) The viral genome copies of CSFV in PK-15 cells at 48 hpi were detected by qPCR; n = 3; *** p < 0.001; **** p < 0.0001. (b) The detection of intracellular cholesterol levels in CSFV-infected PK-15 cells; n = 3; ** p < 0.01; **** p < 0.0001. (c,d) The protein-expression levels of LDLR and PCSK9; n = 3; * p < 0.05. (e,f) The detection of dil-LDL uptake in CSFV-infected PK-15 cells at 48 hpi; n = 3; *** p < 0.001. (g) The inhibition of PCSK9 expression did not affect CSFV replication; n = 3; **** p < 0.0001. (h) The disruption of LDLR did not affect CSFV replication; n = 3. (i,j) The uptake of dil-LDL was completely blocked by the deletion of LDLR; n = 3. (WT, wild-type; LO, ldlr-ko; M, mock; C, CSFV; Rel. prot. lvl, relative protein level; Ic. cholesterol, intracellular cholesterol level; MFI, mean fluorescent intensity).

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  Figure 4. Cholesterol biosynthesis regulates CSFV replication in PK-15 cells. (a,b) The disruption of cholesterol biosynthesis effectively reduced intracellular cholesterol contents and CSFV replication; n = 3; ** p < 0.01; *** p < 0.001; **** p < 0.0001. (c,d) The deficiency of cholesterol biosynthesis upregulated dil-LDL uptake and decreased intracellular cholesterol levels; n = 3; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. (e,f) (WT, wild-type; SO, sqle-ko; HO, hsd17b7-ko; Ic. cholesterol, intracellular cholesterol level; MFI, mean fluorescent intensity).