Journal: Virulence
Article Title: miR-17-5p inhibits classical swine fever virus replication by targeting the PKD2 -regulated AMPK/mTOR autophagy pathway
doi: 10.1080/21505594.2026.2629657
Figure Lengend Snippet: PKD2 regulates autophagic flux via the AMPK/mTOR pathway to promote CSFV proliferation. (A) PK-15 cells were infected with CSFV (MOI = 1), and the expression of autophagy-related markers (LC3I, LC3II, and p62) and CSFV E2 protein was quantified by Western blotting at 48 hours post-infection. (B) quantitative analysis of band intensity was performed using image J software to evaluate autophagy changes induced by CSFV infection. (C) PK-15 cells were transfected with siPKD2-2 for 12 hours. Western blotting was performed to detect the expression of total AMPK, phosphorylated AMPK (Thr172), total mTOR, and phosphorylated mTOR (Ser2448). (D) densitometric quantification of protein bands was carried out using image J software to assess alterations in pathway activity after PKD2 silencing. (E) PK-15 cells were treated with either an AMPK inhibitor (Compound C, 10 μM, 6 hours) or an mTOR inhibitor (Sapanisertib, 100 nM, 6 hours). Cells were then infected with CSFV (MOI = 1), and samples were harvested 24 h post-infection for protein analysis. Western blotting was performed to detect autophagy-related markers (LC3I, LC3II, and p62), key components of the AMPK/mTOR signaling pathway (total and phosphorylated AMPK [Thr172], total and phosphorylated mTOR [Ser2448]), and CSFV E2 protein expression. (F-H) quantitative densitometric analysis of protein bands was conducted using image J software to evaluate alterations in pathway activity and autophagic flux. (I) for viral titer analysis, PK-15 cells were treated with either an AMPK inhibitor (Compound C, 10 μM, 6 hours) or an mTOR inhibitor (Sapanisertib, 100 nM, 6 hours), followed by CSFV inoculation using ten-fold serial dilutions. After 3 days, viral titers were determined using the TCID 50 assay and expressed as Log 10 TCID 50 /0.1mL. Data are presented as mean ± standard deviation (SD), with error bars representing SD from three independent biological replicates. Statistical significance was determined using Student’s t-test and one-way ANOVA. Asterisks denote significant differences compared to control: ns, not significant; * p <0.05; ** p < 0.01; *** p < 0.001. **** p < 0.0001. Representative blots are shown from three independent experiments.
Article Snippet: Anti CSFV E2 mouse mAb , Biorbyt , Or318086.
Techniques: Infection, Expressing, Western Blot, Software, Transfection, Activity Assay, Standard Deviation, Control
![PKD2 regulates autophagic flux via the AMPK/mTOR pathway to promote CSFV proliferation. (A) PK-15 cells were infected with CSFV (MOI = 1), and the expression of autophagy-related markers (LC3I, LC3II, and p62) and CSFV E2 protein was quantified by Western blotting at 48 hours post-infection. (B) quantitative analysis of band intensity was performed using image J software to evaluate autophagy changes induced by CSFV infection. (C) PK-15 cells were transfected with siPKD2-2 for 12 hours. Western blotting was performed to detect the expression of total AMPK, phosphorylated AMPK (Thr172), total mTOR, and phosphorylated mTOR (Ser2448). (D) densitometric quantification of protein bands was carried out using image J software to assess alterations in pathway activity after PKD2 silencing. (E) PK-15 cells were treated with either an AMPK inhibitor (Compound C, 10 μM, 6 hours) or an mTOR inhibitor (Sapanisertib, 100 nM, 6 hours). Cells were then infected with CSFV (MOI = 1), and samples were harvested 24 h post-infection for protein analysis. Western blotting was performed to detect autophagy-related markers (LC3I, LC3II, and p62), key components of the AMPK/mTOR signaling pathway (total and phosphorylated AMPK [Thr172], total and phosphorylated mTOR [Ser2448]), and CSFV E2 protein expression. (F-H) quantitative densitometric analysis of protein bands was conducted using image J software to evaluate alterations in pathway activity and autophagic flux. (I) for viral titer analysis, PK-15 cells were treated with either an AMPK inhibitor (Compound C, 10 μM, 6 hours) or an mTOR inhibitor (Sapanisertib, 100 nM, 6 hours), followed by CSFV inoculation using ten-fold serial dilutions. After 3 days, viral titers were determined using the TCID 50 assay and expressed as Log 10 TCID 50 /0.1mL. Data are presented as mean ± standard deviation (SD), with error bars representing SD from three independent biological replicates. Statistical significance was determined using Student’s t-test and one-way ANOVA. Asterisks denote significant differences compared to control: ns, not significant; * p <0.05; ** p < 0.01; *** p < 0.001. **** p < 0.0001. Representative blots are shown from three independent experiments.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_5864/pmc12915864/pmc12915864__KVIR_A_2629657_F0007_OC.jpg)
