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 is a host target of miR-17-5p involved in the regulation of CSFV replication. (A) venn diagram showing predicted miR-17-5p target genes identified by by TargetScan (1,385 genes) and miRDB (1,333 genes), with 936 overlapping targets. (B) PK-15 cells were transfected with 100 nM miR-17-5p mimics or negative control (miR-NC) for 12h. mRNA levels of candidate target genes were measured by RT-qPCR. (C, D) PK-15 cells were transfected for 12 h with overexpression plasmids for PKD2 (OE- PKD2 ), MED12L (OE- MED12L ), FGD4 (OE- FGD4 ), or control vector, followed by CSFV infection at MOI=1 for 24 h. CSFV RNA and protein levels were analyzed by RT-qPCR (C) and Western blot, (D),respectively, and band intensities were quantified using image J. (E) for viral titer analysis, PK-15 cells were transfected with PKD2 (OE-PKD2), MED12L (OE-MED12L), or FGD4 (OE-FGD4), 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. (F, G) PK-15 cells were transfected with increasing concentrations of miR-17-5p mimics (0–150 nM) for 12 h. Expression of PKD2 at the mRNA and protein levels was evaluated by RT-qPCR (F) and Western blot (G), respectively,the relative intensity of each band was quantified through image J. Data are presented as mean ± standard deviation (SD) from three independent experiments. Statistical significance was determined using Student’s t-test and one-way ANOVA. ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Briefly, PK-15 cells were subjected to the following treatments: BAPTA-AM (10 μM; Sigma-Aldrich, Cat. #A1076) for 6 h to chelate intracellular Ca 2 + ; si PKD2 -2 (100 nM) transfection for 12 h; Sapanisertib (100 nM; MCE, Cat. #HY-13373) for 6 h to inhibit mTOR signaling; Compound C (10 μM; MCE, Cat. #HY-13418A) for 6 h as an AMPK inhibitor; miR-17-5p mimics (100 nM) transfection for 12 h; Co-transfection with miR-17-5p mimics and pcDNA3.1- PKD2 plasmid for 12 h. In some selected groups, CSFV infection was performed at an MOI of 1 following treatment, and cells were harvested at specific time points (e.g. 24 and 48 hpi).

Techniques: Transfection, Negative Control, Quantitative RT-PCR, Over Expression, Control, Plasmid Preparation, Infection, Western Blot, Expressing, Standard Deviation

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: Down-regulation of PKD2 inhibits CSFV replication. (A) PK-15 cells were transfected with siNC, si PKD2 -1, or si PKD2 -2, followed by infection with CSFV at an MOI of 1 after 12 h. Intracellular levels of PKD2 mRNA and CSFV RNA were quantified by RT-qPCR at 0, 24, and 48 hr post-infection. (B) for viral titer analysis, PK-15 cells were transfected with siNC, siPKD2-1, or siPKD2-2, 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.(C) Western blot analysis was performed to assess the expression levels of PKD2 and CSFV E2 at the corresponding time points, and band intensities were quantified using image J. (D) PK-15 cells were transfected with a miR-17-5p mimics or co-transfected with the miR-17-5p mimics and a PKD2 overexpression plasmid (OE- PKD2 ), followed by CSFV infection (MOI = 1) after 12 h. Intracellular CSFV E2 protein levels were analyzed by Western blot, and band intensities were quantified using image J. (E) PK-15 cells were transfected with a miR-17-5p inhibitor or co-transfected with the inhibitor and si PKD2 -2, followed by CSFV infection (MOI=1) after 12 h. CSFV E2 protein expression was examined by Western blot (upper panel), and the relative grayscale intensities were quantified by image J (lower panel). Data are presented as mean ± standard deviation (SD) from three independent experiments. Error bars represent SD. Asterisks denote statistical significance compared with the siNC group (A, B) or Mock group (C, D): ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 (Student’s t-test and one-way ANOVA). Representative blot images from three independent experiments are shown.

Article Snippet: Briefly, PK-15 cells were subjected to the following treatments: BAPTA-AM (10 μM; Sigma-Aldrich, Cat. #A1076) for 6 h to chelate intracellular Ca 2 + ; si PKD2 -2 (100 nM) transfection for 12 h; Sapanisertib (100 nM; MCE, Cat. #HY-13373) for 6 h to inhibit mTOR signaling; Compound C (10 μM; MCE, Cat. #HY-13418A) for 6 h as an AMPK inhibitor; miR-17-5p mimics (100 nM) transfection for 12 h; Co-transfection with miR-17-5p mimics and pcDNA3.1- PKD2 plasmid for 12 h. In some selected groups, CSFV infection was performed at an MOI of 1 following treatment, and cells were harvested at specific time points (e.g. 24 and 48 hpi).

Techniques: Transfection, Infection, Quantitative RT-PCR, Western Blot, Expressing, Over Expression, Plasmid Preparation, Standard Deviation

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 cellular autophagy by modulating intracellular affecting Ca 2+ dynamics. (A) PK-15 cells were transfected with increasing concentrations of si PKD2 (0, 25, 50, 100 nM) for 12 h and then loaded with the calcium-sensitive dye Fura2-AM probe (5 μM). Following chelation of extracellular Ca 2 + with 3 mM EGTA, cells were stimulated with ATP (10 μM), thapsigargin (tg, 1 μM), or ionomycin (iono, 10 μM). Real-time intracellular Ca 2 + flux was measured and expressed as F/F 0 fluorescence intensity ratio. (B) PK-15 cells were treated with the intracellular Ca 2 + chelator BAPTA-AM (6 h) or transfected with si PKD2 -2 (12 h). Western blot analysis was performed to assess LC3-II, LC3-I, and p62 protein levels. The LC3-II/LC3-I ratio and p62 level were quantified using image J. (C) PK-15 cells were transfected with miR-NC, miR-17-5p mimics, or co-transfected with miR-17-5p mimics and a PKD2 overexpression plasmid (OE- PKD2 ), respectively. Protein levels of LC3-II, LC3-I, and p62 were assessed by Western blot. Data are presented as mean ± standard deviation (SD) from three independent experiments. Asterisks denote statistically significant differences compared to the respective control groups: ns, not significant; * p <0.05; ** p <0.01; *** p <0.001 (Student’s t-test and one-way ANOVA). Representative immunoblots from three independent biological replicates are shown.

Article Snippet: Briefly, PK-15 cells were subjected to the following treatments: BAPTA-AM (10 μM; Sigma-Aldrich, Cat. #A1076) for 6 h to chelate intracellular Ca 2 + ; si PKD2 -2 (100 nM) transfection for 12 h; Sapanisertib (100 nM; MCE, Cat. #HY-13373) for 6 h to inhibit mTOR signaling; Compound C (10 μM; MCE, Cat. #HY-13418A) for 6 h as an AMPK inhibitor; miR-17-5p mimics (100 nM) transfection for 12 h; Co-transfection with miR-17-5p mimics and pcDNA3.1- PKD2 plasmid for 12 h. In some selected groups, CSFV infection was performed at an MOI of 1 following treatment, and cells were harvested at specific time points (e.g. 24 and 48 hpi).

Techniques: Transfection, Fluorescence, Western Blot, Over Expression, Plasmid Preparation, Standard Deviation, Control

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: Briefly, PK-15 cells were subjected to the following treatments: BAPTA-AM (10 μM; Sigma-Aldrich, Cat. #A1076) for 6 h to chelate intracellular Ca 2 + ; si PKD2 -2 (100 nM) transfection for 12 h; Sapanisertib (100 nM; MCE, Cat. #HY-13373) for 6 h to inhibit mTOR signaling; Compound C (10 μM; MCE, Cat. #HY-13418A) for 6 h as an AMPK inhibitor; miR-17-5p mimics (100 nM) transfection for 12 h; Co-transfection with miR-17-5p mimics and pcDNA3.1- PKD2 plasmid for 12 h. In some selected groups, CSFV infection was performed at an MOI of 1 following treatment, and cells were harvested at specific time points (e.g. 24 and 48 hpi).

Techniques: Infection, Expressing, Western Blot, Software, Transfection, Activity Assay, Standard Deviation, Control

Journal: International Immunopharmacology

Article Title: Small molecule inhibitor CRT0066101 inhibits cytokine storm syndrome in a mouse model of lung injury

doi: 10.1016/j.intimp.2023.110240

Figure Lengend Snippet: Effect of LPS and CRT0066101 on PKD2 expression and activation in THP-1-derived macrophages. A) THP-1-derived macrophages were treated with different concentrations of LPS (0, 2.5, 5, 10, 20, and 30 ng/mL) for 12 or 24 h, and the phosphorylation levels of PKD2 were detected by western blotting. B) Cells were treated with different concentrations of CRT0066101 (0, 1, 2.5, 5, 10, and 20 μM) in the presence or absence of LPS (20 ng/mL) for 24 h, and the phosphorylation levels of PKD2 were detected by western blotting. Data are presented as the mean ± SEM (n = 3). *** P < 0.001. LPS, lipopolysaccharide; PKD2, protein kinase D2; SEM, standard error of the mean.

Article Snippet: Antibodies against extracellular signal-regulated kinase 1/2 (ERK1/2; cat# 4695), phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (cat# 4370), PKD2 (Cat# 8188), c-Jun N-terminal kinase (JNK; cat# 9252), p-JNK (Thr183/Tyr185) (cat# 9251), horseradish peroxidase (HRP)-linked anti-mouse IgG (cat# 7076) and HRP-linked anti-rabbit IgG (cat# 7074) were purchased from Cell Signaling Technology (Danvers, MA, USA). p-PKD2 (S876) (cat# ab246493), nuclear factor kappa B (NF-κB) P65 (cat# ab32536), p-NF-κB P65 (cat# ab109458) and CD45 (cat# ab40763) were purchased from Abcam (Cambridge, UK).

Techniques: Expressing, Activation Assay, Derivative Assay, Phospho-proteomics, Western Blot

Journal: Cells

Article Title: Epithelial Dynamics of Cystogenesis in Genetic Models of Autosomal Dominant Polycystic Kidney Disease

doi: 10.3390/cells15030297

Figure Lengend Snippet: Lineage tracing of individual Pkd1 or Pkd2 mutant epithelial cells during cyst formation in developmental ADPKD models. ( A ) Confocal images of the cortical and medullary region from kidney sections of CAG CreER ; Rosa Brainbow/ + mice at P 11 following induction with tamoxifen at P 10 . ( B ) Schematic illustration of lineage tracing strategy in the early-onset ADPKD model. Tamoxifen-induced recombination simultaneously inactivates Pkd1 in individual cells and labels them with distinct fluorophores at P 10 ; cystic phenotypes were analyzed at P 24 . ( C ) Representative images of hematoxylin and eosin-stained kidney sections of CAG CreER ; Rosa Brainbow/ + , CAG CreER ; Pkd1 fl/fl ; Rosa Brainbow/ + , and CAG CreER ; Pkd2 fl/fl ; Rosa Brainbow/ + mice at P 24 after a single tamoxifen dose at P 10 . ( D ) Aggregated data of the kidney weight to body weight ratio of the mice. Data represent mean ± SEM; n = 7 mice per group; **** p < 0.0001 by unpaired two-tailed t -test. ( E ) Representative confocal images of renal cortex and medulla of CAG CreER ; Rosa Brainbow/ + , CAG CreER ; Pkd1 fl/fl ; Rosa Brainbow/ + , and CAG CreER ; Pkd2 fl/fl ; Rosa Brainbow/ + mice at P 24 . ( F ) Quantitative analysis of cell numbers in each clone. n = 3 mice per group. ( G ) Percentage distribution (means ± SEM) bar plot of clone size. A total of 891 clones were analyzed in control ( CAG CreER ; Rosa Brainbow/ + ) kidneys, and 513 clones each in Pkd1 and Pkd2 knockouts. Scale bars: ( A , E ): 50 μm. ( C ): 1 mm.

Article Snippet: Pkd2 fl mice was generated by Cyagen using CRISPR/CAS9-mediated genome editing to insert loxP sites flanking exons 4–5, enabling Cre-mediated deletion.

Techniques: Mutagenesis, Staining, Two Tailed Test, Clone Assay, Control

Journal: Cells

Article Title: Epithelial Dynamics of Cystogenesis in Genetic Models of Autosomal Dominant Polycystic Kidney Disease

doi: 10.3390/cells15030297

Figure Lengend Snippet: Lineage tracing of Pkd1 or Pkd2 mutant renal epithelial cells during cyst formation in adult-onset ADPKD models. ( A ) Confocal images of the cortical and medullary regions of kidney sections of CAG CreER ; Rosa Brainbow/ + mice at P 32 induced with tamoxifen at P 28 . ( B ) Schematic illustration of lineage tracing strategy used to follow Pkd1 or Pkd2 mutant cells during cystogenesis in an adult-onset renal cyst model. Tamoxifen was administered at P 28 , and phenotypes were analyzed at 2, 4, and 6 months. ( C ) Representative confocal images of cortical and medullary regions from the indicated genotypes at 2 months. ( D ) Quantitative analysis of cell numbers in each clone. n = 3 mice per group. The percentage (means ± SEM) distribution of clone sizes in CAG CreER ; Rosa Brainbow/ + , CAG CreER ; Pkd1 fl/fl ; Rosa Brainbow/ + , and CAG CreER ; Pkd2 fl/fl ; Rosa Brainbow/ + mice. A total of 891 clones were analyzed in control ( CAG CreER ; Rosa Brainbow/ + ) kidneys and 513 clones in each Pkd1 and Pkd2 mutant group. ( E ) Representative confocal images of the cortex and medulla regions of kidney sections in mice at 4 months. ( F ) Quantitative analysis of cell numbers in each clone. n = 3 mice per group. The percentage (means ± SEM) distribution of clone sizes in CAG CreER ; Rosa Brainbow/ + , CAG CreER ; Pkd1 fl/fl ; Rosa Brainbow/ + , and CAG CreER ; Pkd2 fl/fl ; Rosa Brainbow/ + mice. A total of 891 clones were analyzed in control ( CAG CreER ; Rosa Brainbow/ + ) and 291 in each Pkd1 and Pkd2 mutant group. ( G ) Representative confocal images of the cortical and medullary regions of kidney sections of mice at 6 months. ( H ) Quantitative analysis of cell numbers in each clone. n = 3 mice per group. The percentage (means ± SEM) distribution of clone sizes in CAG CreER ; Rosa Brainbow/ + , CAG CreER ; Pkd1 fl/fl ; Rosa Brainbow/ + , and CAG CreER ; Pkd2 fl/fl ; Rosa Brainbow/ + mice. A total of 891 clones were analyzed in control ( CAG CreER ; Rosa Brainbow/ + ) and 291 in Pkd1 and Pkd2 mutant groups. Scale bar: ( A , C , E , G ): 50 μm.

Article Snippet: Pkd2 fl mice was generated by Cyagen using CRISPR/CAS9-mediated genome editing to insert loxP sites flanking exons 4–5, enabling Cre-mediated deletion.

Techniques: Mutagenesis, Clone Assay, Control

Journal: Cells

Article Title: Epithelial Dynamics of Cystogenesis in Genetic Models of Autosomal Dominant Polycystic Kidney Disease

doi: 10.3390/cells15030297

Figure Lengend Snippet: Lineage tracing of individual cholangiocytes for cyst formation in ADPKD model. ( A ) Representative confocal images of liver sections from CK19 CreER ; Rosa Brainbow/ + mice at P 32 following tamoxifen induction at P 28 . ( B ) Schematic illustrating the lineage tracing strategy for bile duct cells in ADPKD. Mice were induced with tamoxifen at P 28 , and phenotypes were analyzed at 2, 4, and 6 months. ( C ) Representative H&E-stained liver sections from CK19 CreER ; Pkd1 fl/ + ; Rosa Brainbow/ + and CK19 CreER ; Pkd1 fl/fl ; Rosa Brainbow/ + mice at 2, 4, and 6 months. Mice were induced with tamoxifen at P 28 . ( D ) Aggregated data of liver weight to body weight ratio of CK19 CreER ; Pkd1 fl/ + ; Rosa Brainbow/ + and CK19 CreER ; Pkd1 fl/fl ; Rosa Brainbow/ + mice at 2, 4, and 6 months. Mice were induced with tamoxifen at P 28 . Data are represented as mean ± SEM; n = 7 mice per group; ns (no significant), *** p < 0.001, **** p < 0.0001 by unpaired two-tailed t -test. ( E ) Representative confocal images of liver sections from CK19 CreER ; Pkd1 fl/ + ; Rosa Brainbow/ + ; and CK19 CreER ; Pkd1 fl/fl ; Rosa Brainbow/ + mice at 2, 4 and 6 months. ( F ) Quantification of the cell numbers per clone and the percentage (means ± SEM) distribution of clone sizes in cholangiocytes at 2 ( F ), 4 ( G ), and 6 ( H ) months. n = 3 mice per group. A total of 375 clones were analyzed in control ( CK19 CreER ; Pkd1 fl/ + ; Rosa Brainbow/ + ) mice versus 363 clones in CK19 CreER ; Pkd1 fl/fl ; Rosa Brainbow/ + mice at 2 months, 456 vs. 228 at 4 months, and 387 versus 228 at 6 months. Scale bar: ( A , E ): 50 μm. ( C ): 200 μm.

Article Snippet: Pkd2 fl mice was generated by Cyagen using CRISPR/CAS9-mediated genome editing to insert loxP sites flanking exons 4–5, enabling Cre-mediated deletion.

Techniques: Staining, Two Tailed Test, Clone Assay, Control