llc pk1 cells  (ATCC)

Journal: Journal of Virology

Article Title: PEX19 restricts porcine deltacoronavirus replication through farnesylation-dependent and -independent mechanisms

doi: 10.1128/jvi.02097-25

Figure Lengend Snippet: PEX19 exhibits stronger anti-PDCoV activity. ( A and B ) LLC-PK1 cells were transfected with plasmids encoding PEX5, PEX7, PEX11α, PEX11β, PEX11γ, PEX12, PEX14, PEX16, PEX19, or an empty vector as control. At 12 h post-transfection, cells were infected with PDCoV at a multiplicity of infection (MOI) of 1. At 12 h post-infection, cell lysates and supernatants were harvested to assess viral replication by RT-qPCR ( A ) and TCID₅₀ assay ( B ). ( C and D ) Dose-dependent inhibition of PDCoV replication by PEX19. LLC-PK1 cells were transfected with increasing amounts (0, 0.5, 1.0, and 2.0 μg) of PEX19 expression plasmid. At 12 h post-transfection, cells were infected with PDCoV (MOI=1). Samples were harvested at 12 h post-infection for viral RNA quantification by RT-qPCR ( C ) and viral titration by TCID 50 assay ( D ). ( E and F ) Generation and validation of PEX19 knockout (KO) cells. PEX19 expression was analyzed by immunofluorescence assay (scale bar, 5 μm). ( E ) and Western blotting ( F ) using a rabbit polyclonal antibody against PEX19. ( G and H ) Effects of PEX19 knockout and complementation on PDCoV replication. PEX19 KO cells were transfected with either the PEX19 expression plasmid or the empty vector. WT LLC-PK1 cells, which were transfected with an empty vector, were used as controls. At 12 h post-transfection, cells were infected with PDCoV (MOI = 1), and samples were collected 12 h later for RT-qPCR ( G ) and TCID 50 assay ( H ). Values are shown as means ± SD. Statistical analyses for panels A to D, G, and H were performed with one-way ANOVA. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: LLC porcine kidney (LLC-PK1) cells were purchased from the American Type Culture Collection (ATCC CL-101; Manassas, VA, USA).

Techniques: Activity Assay, Transfection, Plasmid Preparation, Control, Infection, Quantitative RT-PCR, Inhibition, Expressing, Titration, Biomarker Discovery, Knock-Out, Immunofluorescence, Western Blot

Journal: Journal of Virology

Article Title: PEX19 restricts porcine deltacoronavirus replication through farnesylation-dependent and -independent mechanisms

doi: 10.1128/jvi.02097-25

Figure Lengend Snippet: Farnesylation of PEX19 is required for its antiviral activity against PDCoV. ( A ) Schematic diagram of PEX19 structure and farnesylation. ( B and C ) FTI-277 inhibits the antiviral activity of PEX19. Cells were transfected with a PEX19-expressing plasmid or vector control. At 12 h post-transfection, cells were infected with PDCoV at an MOI of 1. At 6 h post-infection (hpi), the culture medium was replaced with fresh medium containing 20 μM FTI-277 or DMSO (vehicle control), and incubation continued for an additional 6 h. Samples were collected at 12 hpi and analyzed by RT-qPCR ( B ) and TCID 50 assay ( C ). ( D ) Construction of PEX19 farnesylation-deficient mutants. Two PEX19 mutants were generated via site-directed mutagenesis: PEX19 C268S (cysteine at position 268 mutated to serine) and PEX19 ΔCaaX (deletion of the CaaX motif). ( E–G ) Effects of WT and mutant PEX19 on PDCoV replication in LLC-PK1 cells. Cells were transfected with plasmids encoding WT PEX19, PEX19 C268S, or PEX19 ΔCaaX. At 12 h post-transfection, cells were infected with PDCoV (MOI=1). Samples were collected after 12 h post-infection and analyzed by RT-qPCR ( E ), TCID 50 assay ( F ), and Western blot analysis ( G ). Cells transfected with an empty vector were used as negative controls. ( H–J ) Effects of PEX19 WT and mutants on PDCoV replication in PEX19 KO cells. PEX19 KO cells were transfected with plasmids expressing WT or mutant PEX19. At 12 h post-transfection, cells were infected with PDCoV (MOI = 1) and harvested 12 h later for RT-qPCR ( H ), TCID 50 assay ( I ), and Western blot analysis ( J ). WT LLC-PK1 cells served as a control. β-actin was used as a loading control. Values are shown as means ± SD. Statistical analyses for panels B, C, E, F, H, and I were performed with one-way ANOVA. NS., no significance; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: LLC porcine kidney (LLC-PK1) cells were purchased from the American Type Culture Collection (ATCC CL-101; Manassas, VA, USA).

Techniques: Activity Assay, Transfection, Expressing, Plasmid Preparation, Control, Infection, Incubation, Quantitative RT-PCR, Generated, Mutagenesis, Western Blot

Journal: Journal of Virology

Article Title: PEX19 restricts porcine deltacoronavirus replication through farnesylation-dependent and -independent mechanisms

doi: 10.1128/jvi.02097-25

Figure Lengend Snippet: Cholesterol positively regulates PDCoV replication. ( A ) Cytotoxicity of methyl-β-cyclodextrin (MβCD) on LLC-PK1 cells was evaluated using the CCK-8 assay. Cells were treated with increasing concentrations of MβCD (0, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, and 20 mM) for 24 h prior to analysis. ( B ) Cholesterol depletion by MβCD was assessed using the Amplex Red Cholesterol Assay Kit. LLC-PK1 cells were incubated with 0, 5, 10, or 15 mM MβCD for 1 h, and total cholesterol levels were subsequently measured. ( C–E ) Effects of cholesterol depletion on PDCoV replication in PEX19 KO cells. WT and PEX19 KO cells were treated with the indicated concentrations of MβCD (0, 5, 10, or 15 mM) at 37°C for 1 h, followed by infection with PDCoV at an MOI of 1. At 12 h post-infection, viral replication was assessed by RT-qPCR ( C ), TCID₅₀ assay ( D ), and Western blot analysis ( E ). ( F–H ) Restoration of cholesterol rescues PDCoV replication. WT and PEX19 KO cells were pretreated with 10 mM MβCD at 37°C for 1 h, followed by supplementation with water-soluble cholesterol (400 μg/mL) for 1 h. Cells were then infected with PDCoV (MOI = 1), and samples were collected at 12 h post-infection for RT-qPCR ( F ), TCID₅₀ assay ( G ), and Western blot analysis ( H ). β-actin was used as a loading control. Values are shown as means ± SD. Statistical analyses for panels B, C, D, F, and G were performed with one-way ANOVA. NS., no significance; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: LLC porcine kidney (LLC-PK1) cells were purchased from the American Type Culture Collection (ATCC CL-101; Manassas, VA, USA).

Techniques: CCK-8 Assay, Amplex Red Cholesterol Assay, Incubation, Infection, Quantitative RT-PCR, Western Blot, Control

Journal: Journal of Virology

Article Title: PEX19 restricts porcine deltacoronavirus replication through farnesylation-dependent and -independent mechanisms

doi: 10.1128/jvi.02097-25

Figure Lengend Snippet: PEX19 promotes the degradation of PDCoV nsp2. ( A and B ) PEX19 reduces the protein levels of PDCoV nsp2. LLC-PK1 cells were co-transfected with pCAGGS-Flag-PEX19 and pCAGGS-HA-nsp2. At 24 h post-transfection, cells were lysed and supernatants collected. A small portion of each lysate was reserved as whole-cell lysate (WCL), and the remaining lysates were subjected to coimmunoprecipitation (Co-IP) using anti-HA ( A ) or anti-Flag ( B ) monoclonal antibodies. ( C ) Farnesylation of PEX19 is required for degradation of PDCoV nsp2. LLC-PK1 cells cultured in 6-well plates were co-transfected with 1 μg of pCAGGS-HA-nsp2 and increasing amounts (0, 0.25, 0.5, and 1 μg) of WT PEX19, PEX19 C268S, or PEX19 ΔCaaX expression constructs. Western blotting was performed using anti-HA, anti-Flag, and anti-β-actin antibodies, respectively. ( D ) FTI-277 abrogates PEX19-mediated degradation of PDCoV nsp2. LLC-PK1 cells were co-transfected with plasmids encoding HA-PDCoV nsp2 and Flag-PEX19. At 18 h post-transfection, cells were treated with 20 μM FTI-277 or DMSO for 6 h. Cells were then harvested for Western blot analysis using anti-HA, anti-Flag, and anti-β-actin antibodies. ( E ) Analysis of the PEX19-mediated degradation pathway of PDCoV nsp2. LLC-PK1 cells were co-transfected with pCAGGS-HA-nsp2 and pCAGGS-Flag-PEX19 or an empty vector. At 12 h post-transfection, cells were treated with DMSO, MG132 (10 μM), Z-VAD (20 μM), chloroquine (CQ, 5 μM), or NH₄Cl (10 mM) for 12 h. Cell lysates were analyzed by Western blotting. ( F and G ) PEX19-mediated degradation of PDCoV nsp2 is impaired in autophagy-deficient cells. LLC-PK1 cells were transfected with siRNA specific to ATG5 ( F ) or ATG7 ( G ). After 24 h, the cells were subsequently co-transfected with pCAGGS-HA-nsp2 and increasing amounts of pCAGGS-Flag-PEX19. Following an additional 24 h, the cells were harvested, and the lysates were analyzed by Western blot using anti-HA and anti-Flag antibodies to detect nsp2 and PEX19, respectively. β-actin was used as a loading control.

Article Snippet: LLC porcine kidney (LLC-PK1) cells were purchased from the American Type Culture Collection (ATCC CL-101; Manassas, VA, USA).

Techniques: Transfection, Co-Immunoprecipitation Assay, Bioprocessing, Cell Culture, Expressing, Construct, Western Blot, Plasmid Preparation, Control

Journal: Journal of Virology

Article Title: PEX19 restricts porcine deltacoronavirus replication through farnesylation-dependent and -independent mechanisms

doi: 10.1128/jvi.02097-25

Figure Lengend Snippet: PEX19 induces low-level IFN responses independent of its farnesylation. ( A and B ) Activation of IFN promoters by PEX19. LLC-PK1 cells were co-transfected with IFN-β-Luc or IFN-λ1-Luc reporter plasmids and pRL-TK together with increasing amounts of pCAGGS-Flag-PEX19. Luciferase activity was measured at 24 h post-transfection using a dual-luciferase reporter assay system. ( C and D ) Endogenous IFN induction by PEX19. LLC-PK1 cells were transfected with increasing amounts of pCAGGS-Flag-PEX19, and mRNA levels of IFN-β ( C ) and IFN-λ1 ( D ) were determined by RT-qPCR at 24 h post-transfection. ( E and F ) Induction of IFN by PEX19 assessed by VSV-GFP bioassay. LLC-PK1 cells were transfected with increasing amounts of pCAGGS-Flag-PEX19. At 24 h post-transfection, cell culture supernatants were collected. HEK-293T cells were infected with VSV-GFP (MOI = 0.01) for 1 h, followed by treatment with the collected supernatants for 8 h. ( E ) Viral replication was visualized by fluorescence microscopy. Representative images show VSV-GFP (green) and cell nuclei stained with DAPI (blue). Scale bar, 20 μm. ( F ) Quantification of the mean fluorescence intensity (MFI) of VSV-GFP corresponding to ( E ) using Image J software. ( G and H ) Farnesylation-independent induction of IFN by PEX19. PEX19 KO cells were transfected with empty vector, WT PEX19, PEX19 C268S, or PEX19 ΔCaaX constructs. LLC-PK1 WT cells were used as a control. The mRNA levels of IFN-β ( G ) and IFN-λ1 ( H ) were measured by RT-qPCR. ( I and J ) Farnesylation-independent induction of IFN by PEX19. PEX19 KO LLC-PK1 cells were transfected with the indicated constructs. Culture supernatants were harvested at 24 h post-transfection. HEK-293T cells were infected with VSV-GFP (MOI = 0.01) for 1 h and subsequently treated with the supernatants for 12 h. ( I ) Representative images show VSV-GFP (green) and cell nuclei stained with DAPI (blue). Scale bar, 20 μm. ( J ) Quantification of VSV-GFP fluorescence intensity corresponding to ( I ) using Image J software. ( K and L ) IFN response to PDCoV infection in LLC-PK1 WT cells and PEX19 KO cells. LLC-PK1 WT cells or PEX19 KO cells were infected with PDCoV (MOI=1). Total RNA was extracted at 12 h post-infection, and IFN-β ( K ) and IFN-λ1 ( L ) mRNA levels were analyzed by RT-qPCR. ( M ) Peroxisome morphology in LLC-PK1 WT cells and PEX19 KO cells. Cells were fixed, and IFA was performed with antibodies against the peroxisomal membrane protein PMP70 (red), and cell nuclei were counterstained with DAPI (blue). Fluorescence was visualized by using a confocal laser scanning microscope. Scale bar, 5 μm.Values are shown as means ± SD. Statistical analyses were performed with one-way ANOVA (for panels A to D, F to H, and J) or two-way ANOVA (for panels K and L) . ns., no significance; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: LLC porcine kidney (LLC-PK1) cells were purchased from the American Type Culture Collection (ATCC CL-101; Manassas, VA, USA).

Techniques: Activation Assay, Transfection, Luciferase, Activity Assay, Reporter Assay, Quantitative RT-PCR, Bioassay, Cell Culture, Infection, Fluorescence, Microscopy, Staining, Software, Plasmid Preparation, Construct, Control, Membrane, Laser-Scanning Microscopy