pk15 cells  (ATCC)

Journal: Veterinary Sciences

Article Title: Identification of the Role of NAT10 in the Regulation of Porcine Circovirus Type 2 Infection

doi: 10.3390/vetsci12121160

Figure Lengend Snippet: Construction of PCV2 infection model (MOI = 1) in PK15 cells. ( a ) Temporal profile of relative Cap mRNA expression in PK15 cells following PCV2 infection at indicated time intervals (0, 12, 24, 36, 48, and 72 h). ( b ) Cap protein abundance in PCV2-challenged PK15 cells across different time courses. Values represent mean ± SD from triplicate experiments, * p < 0.05, ** p < 0.01.

Article Snippet: The PK15 cell line (ATCC, CCL-33) was propagated in DMEM (Thermo, Waltham, MA, USA) enriched with 10% FBS (Bio-Channel, Nanjing, China) and 100 U/mL penicillin–streptomycin solution (Solarbio, Beijing, China).

Techniques: Infection, Expressing, Quantitative Proteomics

Journal: Veterinary Sciences

Article Title: Identification of the Role of NAT10 in the Regulation of Porcine Circovirus Type 2 Infection

doi: 10.3390/vetsci12121160

Figure Lengend Snippet: NAT10 expression in PK15 cells after PCV2 infection. ( a ) Relative NAT10 mRNA expression was analyzed by qRT-PCR at 24 h and 48 h post-treatment compared with the mock control (normal PK15 cells) group. ( b ) Western blot analysis of Cap and NAT10 protein levels at the indicated time points. HSP90 was used as an internal control. Values represent mean ± SD from triplicate experiments, ** p < 0.01.

Article Snippet: The PK15 cell line (ATCC, CCL-33) was propagated in DMEM (Thermo, Waltham, MA, USA) enriched with 10% FBS (Bio-Channel, Nanjing, China) and 100 U/mL penicillin–streptomycin solution (Solarbio, Beijing, China).

Techniques: Expressing, Infection, Quantitative RT-PCR, Control, Western Blot

Journal: Veterinary Sciences

Article Title: Identification of the Role of NAT10 in the Regulation of Porcine Circovirus Type 2 Infection

doi: 10.3390/vetsci12121160

Figure Lengend Snippet: NAT10 is essential for regulating PCV2 replication in PK15 cells. ( a ) The silencing efficacy of NAT10 in PK15 cells was assessed via quantitative PCR analysis. ( b ) Relative Cap mRNA expresssion following NAT10 depletion (si-NAT10-161) was quantified by qPCR. ( c ) Cap protein levels after NAT10 knockdown (si-NAT10-161) were analyzed through Western blotting. Two lanes for siNC and siNAT10 represent two replicates. ( d ) Indirect immunofluorescence staining: DAPI (blue) labels cell nuclei; anti-PCV2 Cap antibody (green) marks viral Cap protein. Images were captured using fluorescence microscopy (magnification: 100×, scale bar = 100 µm). Values represent mean ± SD from triplicate experiments, ** p < 0.01.

Article Snippet: The PK15 cell line (ATCC, CCL-33) was propagated in DMEM (Thermo, Waltham, MA, USA) enriched with 10% FBS (Bio-Channel, Nanjing, China) and 100 U/mL penicillin–streptomycin solution (Solarbio, Beijing, China).

Techniques: Real-time Polymerase Chain Reaction, Knockdown, Western Blot, Immunofluorescence, Staining, Fluorescence, Microscopy

Journal: Veterinary Sciences

Article Title: Identification of the Role of NAT10 in the Regulation of Porcine Circovirus Type 2 Infection

doi: 10.3390/vetsci12121160

Figure Lengend Snippet: Transcriptomic profiling comparing siNAT10 and siNC samples. ( a ) Volcano diagram depicting the pattern of modulated genes. The horizontal axis displays log 2 fold values, while the vertical axis shows −log 10 ( p -value). Genes exhibiting increased expression appear in red, those with decreased expression in blue, and unchanged genes in gray. siNAT10 represents the PK15 cells with si-NAT10-161 vector. ( b ) Color-coded matrix presenting unsupervised clustering of DEGs across siNAT10 and siNC groups. Horizontal entries correspond to individual genes; vertical entries represent independent biological replicates. Red and blue hues indicate elevated and reduced transcript abundance, respectively.

Article Snippet: The PK15 cell line (ATCC, CCL-33) was propagated in DMEM (Thermo, Waltham, MA, USA) enriched with 10% FBS (Bio-Channel, Nanjing, China) and 100 U/mL penicillin–streptomycin solution (Solarbio, Beijing, China).

Techniques: Expressing, Plasmid Preparation

Journal: Veterinary Sciences

Article Title: Identification of the Role of NAT10 in the Regulation of Porcine Circovirus Type 2 Infection

doi: 10.3390/vetsci12121160

Figure Lengend Snippet: Potential targets identification of NAT10 in PCV2 infection. ( a ) Protein–Protein Interaction (PPI) network generated from differentially modulated genes. Network nodes symbolize proteins, and connecting lines symbolize protein–protein interaction. Gene expression status is indicated by node color: green for decreased expression and red for increased expression. ( b ) Venn Diagram of differentially expressed genes in PCV2-infected kidney tissues and NAT10-knockdown PK15 cells. ( c ) qPCR results quantifying the relative NR1H4 mRNA expression in different treatment groups (PCV2 vs. MOCK, siNAT10 vs. siNC). ( d ) Western blot analysis of NR1H4 protein. Two lanes for each represent two replicates. Values represent mean ± SD from triplicate experiments, * p < 0.05, ** p < 0.01.

Article Snippet: The PK15 cell line (ATCC, CCL-33) was propagated in DMEM (Thermo, Waltham, MA, USA) enriched with 10% FBS (Bio-Channel, Nanjing, China) and 100 U/mL penicillin–streptomycin solution (Solarbio, Beijing, China).

Techniques: Infection, Generated, Gene Expression, Expressing, Knockdown, Western Blot

Journal: Frontiers in Microbiology

Article Title: PCV2 Cap protein nuclear import via importin α/β receptor: molecular insights and antiviral potential

doi: 10.3389/fmicb.2025.1701697

Figure Lengend Snippet: The nuclear import mechanism of C3-GST-Cap. (A) PK15 cells co-transfected with plasmids pEGFP-Cap-GST with pmCherry-C1, pmCherry-C1-Ran, pmCherry-C1-RanQ69L, pmCherry-C1-M9M, pmCherry-C1-Bimax2 for 24 h. Green fluorescence indicates cells expressing pEGFP-Cap-GST, while red fluorescence signifies cells expressing pmCherry fusion proteins. All photomicrographs were captured at a magnification of 400×, with a scale bar of 50 μM. (B) The Cap fluorescence ratio of nucleus to cytoplasm (N/C) was calculated for the selected regions of interest basing on each confocal microscopy image by ImageJ software, and 40 cells were chosen for statistical analysis under each condition, Data are presented as means ± SEM of three independent biological experiments. Statistical analysis was conducted using Student’s t -test. (C) The graph illustrates the percentages of cells with exclusive nuclear (N) distribution or with both cytoplasmic and nuclear (C + N) distribution of pEGFP-Cap-GST, based on the distribution of Cap in various treatment groups as shown in panel (A) .

Article Snippet: The porcine kidney epithelial cell line (PK15) (CCL-33, ATCC, Manassas, VA, United States) and the human embryonic kidney epithelial cell line (HEK 293 T) (CRL-3216, ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Servicebio) supplemented with 10% fetal bovine serum.

Techniques: Transfection, Fluorescence, Expressing, Confocal Microscopy, Software

Journal: Frontiers in Microbiology

Article Title: PCV2 Cap protein nuclear import via importin α/β receptor: molecular insights and antiviral potential

doi: 10.3389/fmicb.2025.1701697

Figure Lengend Snippet: The nuclear import mechanism of Myc-Cap. (A) PK15 cells co-transfected with plasmids Myc-Cap with pmCherry-C1, pmCherry-C1-Ran, pmCherry-C1-RanQ69L, pmCherry-C1-M9M, pmCherry-C1-Bimax2 for 24 h. The resultant cells were fixed, incubated with mouse anti-Cap antibody, and then stained with anti-mouse FITC. Green fluorescence indicates cells expressing Myc-Cap, while red fluorescence signifies cells expressing pmCherry fusion proteins. All images were captured using the parameters specified in . Scale bar, 50 μm. (B) The fluorescence ratio of nucleus to cytoplasm (N/C) was measured utilizing the same methodology detailed in , N = 40 cells for each condition. Data are presented as means ± SEM of three independent biological experiments. (C) The graph illustrates the percentages of cells with exclusive nuclear (N) distribution or with both cytoplasmic and nuclear (C + N) distribution of Myc-Cap, based on the distribution of Cap in various treatment groups as shown in panel (A) .

Article Snippet: The porcine kidney epithelial cell line (PK15) (CCL-33, ATCC, Manassas, VA, United States) and the human embryonic kidney epithelial cell line (HEK 293 T) (CRL-3216, ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Servicebio) supplemented with 10% fetal bovine serum.

Techniques: Transfection, Incubation, Staining, Fluorescence, Expressing

Journal: Frontiers in Microbiology

Article Title: PCV2 Cap protein nuclear import via importin α/β receptor: molecular insights and antiviral potential

doi: 10.3389/fmicb.2025.1701697

Figure Lengend Snippet: Importazole (IPZ) blocks Importin-β-dependent nuclear localization of Cap and inhibits the production of infectious PCV2 progeny. (A) The viability of PK15 cells pretreated with 10, 20, or 40 μM of importazole was analyzed using a CCK-8 assay. (B) Confocal dish-cultured PK15 cells were transfected with pEGFP-GST-Cap or Myc-Cap. At 8 h post-transfection, cells were treated with dimethyl sulfoxide (DMSO) or IPZ (20 μM). The cells were fixed at 24 h post-transfection. The distribution of Cap was detected with an anti-Cap antibody and observed by confocal microscopy. Scale bar, 50 μm. (C) The methodology used in this panel was the same as , . (D) The graph depicts the percentages of cells exhibiting exclusive nuclear (N) localization or a combination of cytoplasmic and nuclear (C + N) localization of pEGFP-Cap-GST or Myc-Cap, based on the distribution of Cap within various treatment groups as depicted in panel (B) . PK15 cells were infected with PCV2 at an MOI of 1 and treated with the indicated concentration of IPZ, cells were harvested at 24 hpi. (E) Viral protein synthesis was assayed by western blotting using antibodies against the Cap protein, with β-actin serving as a loading control. WB bands were quantified using densitometry by ImageJ software and normalized against β-actin. The data are presented as means ± standard deviations (SD). Statistical analysis was conducted using Student’s t -test. (F) The resultant cells were fixed, incubated with mouse anti-Cap antibody, and then stained with anti-mouse FITC. All images were captured using the parameters specified in . Scale bar, 50 μm. (G) The fluorescence ratio of nucleus to cytoplasm (N/C) was measured as . (H) PK15 cells were infected with PCV2 at a multiplicity of infection (MOI) of 1 and treated with the indicated concentration of IPZ for 12 h. Cells were harvested at 24 hpi, and viral titers were determined. The results are presented as the mean TCID50 ± standard deviation (SD) ( n = 3). Three independent biological experiments were performed.

Article Snippet: The porcine kidney epithelial cell line (PK15) (CCL-33, ATCC, Manassas, VA, United States) and the human embryonic kidney epithelial cell line (HEK 293 T) (CRL-3216, ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Servicebio) supplemented with 10% fetal bovine serum.

Techniques: CCK-8 Assay, Cell Culture, Transfection, Confocal Microscopy, Infection, Concentration Assay, Western Blot, Control, Software, Incubation, Staining, Fluorescence, Standard Deviation

Journal: Frontiers in Microbiology

Article Title: PCV2 Cap protein nuclear import via importin α/β receptor: molecular insights and antiviral potential

doi: 10.3389/fmicb.2025.1701697

Figure Lengend Snippet: Ivermectin (IVM) inhibits Imp-α-mediated nuclear localization of Cap but does not affect the production of infectious PCV2 progeny. (A) The viability of PK15 cells pretreated with 10, 15, or 25 μM of IVM was analyzed using a CCK-8 assay. (B) Confocal dish-cultured PK15 cells were transfected with pEGFP-Cap-GST or Myc-Cap. At 8 h post-transfection, cells were treated with dimethyl sulfoxide (DMSO) or IVM (15 μM). The cells were fixed at 24 h post-transfection. The distribution of Cap was detected with an anti-Cap antibody and observed by confocal microscopy. Scale bar, 50 μm. (C) The methodology used in this panel was the same as , . (D) The graph shows the percentages of cells displaying exclusive nuclear (N) localization or a combination of cytoplasmic and nuclear (C + N) localization for pEGFP-Cap-GST or Myc-Cap. The classification is based on the distribution of Cap across various treatment groups, as depicted in Panel B. PK15 cells were infected with PCV2 at an MOI of 1 and treated with the indicated concentration of IVM, cells were harvested at 24 hpi. (E) Viral protein synthesis was assayed by western blotting (WB) using antibodies against the Cap protein, with β-actin serving as a loading control. The WB bands were quantified as shown in . The data are presented as means ± standard deviations (SD). Statistical analysis was conducted using Student’s t -test. (F) The resultant cells were fixed and treated as shown in . (G) The fluorescence ratio of nucleus to cytoplasm (N/C) was measured as . (H) PK15 cells were infected with PCV2 and treated with IVM at a concentration of 15 μM for 12 h. Subsequently, the cells were harvested, and viral titers were detected, as indicated in .

Article Snippet: The porcine kidney epithelial cell line (PK15) (CCL-33, ATCC, Manassas, VA, United States) and the human embryonic kidney epithelial cell line (HEK 293 T) (CRL-3216, ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Servicebio) supplemented with 10% fetal bovine serum.

Techniques: CCK-8 Assay, Cell Culture, Transfection, Confocal Microscopy, Infection, Concentration Assay, Western Blot, Control, Fluorescence