swine kidney cell line pk 15  (ATCC)


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    ATCC swine kidney cell line pk 15
    FMDV infection-induced autophagy plays an important role in viral replication. ( A ) <t>PK-15</t> cells were mock infected or infected with FMDV (MOI = 1) for 0, 0.5, 1, 1.5, 2 and 3 h. The expression of LC3B and SQSTM1 was analyzed by western blot. The level of 3D was analyzed by RT-PCR. ACTB and GAPDH were used as a sample loading control. ( B ) The cells were then fixed and processed for indirect immunofluorescence using antibodies against LC3B and the 3D protein, followed by the corresponding secondary antibodies. The fluorescence signals were visualized by confocal immunofluorescence microscopy. ( C ) ATG5 knockdown (KD) and wild-type cells were infected with FMDV (MOI = 1) for 2 and 3 h. The expression of ATG5 and LC3B was analyzed by western blot. ( D and E ) ATG5 KD and wild-type cells were infected with FMDV (MOI = 1). At 3 hpi, both the extracellular and intracellular copy numbers of FMDV were measured by qRT-PCR; both the extracellular and intracellular virus titers were measured by TCID 50 . The data represent the mean ± SD of 3 independent experiments. ***P < 0.001. ( F ) PK-15 cells were mock infected or infected with FMDV (MOI = 1) for 6 and 9 h. The expression of SQSTM1 and VP1 were analyzed by western blot. ( G ) Cells were transfected with pmCherry-GFP-LC3B for 24 h, followed by FMDV infection (MOI = 1) and treatment with Baf-A1. The fluorescence signals were visualized by confocal immunofluorescence microscopy. Scale bars: 10 μm.
    Swine Kidney Cell Line Pk 15, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Foot-and-mouth disease virus capsid protein VP2 activates the cellular EIF2S1-ATF4 pathway and induces autophagy via HSPB1"

    Article Title: Foot-and-mouth disease virus capsid protein VP2 activates the cellular EIF2S1-ATF4 pathway and induces autophagy via HSPB1

    Journal: Autophagy

    doi: 10.1080/15548627.2017.1405187

    FMDV infection-induced autophagy plays an important role in viral replication. ( A ) PK-15 cells were mock infected or infected with FMDV (MOI = 1) for 0, 0.5, 1, 1.5, 2 and 3 h. The expression of LC3B and SQSTM1 was analyzed by western blot. The level of 3D was analyzed by RT-PCR. ACTB and GAPDH were used as a sample loading control. ( B ) The cells were then fixed and processed for indirect immunofluorescence using antibodies against LC3B and the 3D protein, followed by the corresponding secondary antibodies. The fluorescence signals were visualized by confocal immunofluorescence microscopy. ( C ) ATG5 knockdown (KD) and wild-type cells were infected with FMDV (MOI = 1) for 2 and 3 h. The expression of ATG5 and LC3B was analyzed by western blot. ( D and E ) ATG5 KD and wild-type cells were infected with FMDV (MOI = 1). At 3 hpi, both the extracellular and intracellular copy numbers of FMDV were measured by qRT-PCR; both the extracellular and intracellular virus titers were measured by TCID 50 . The data represent the mean ± SD of 3 independent experiments. ***P < 0.001. ( F ) PK-15 cells were mock infected or infected with FMDV (MOI = 1) for 6 and 9 h. The expression of SQSTM1 and VP1 were analyzed by western blot. ( G ) Cells were transfected with pmCherry-GFP-LC3B for 24 h, followed by FMDV infection (MOI = 1) and treatment with Baf-A1. The fluorescence signals were visualized by confocal immunofluorescence microscopy. Scale bars: 10 μm.
    Figure Legend Snippet: FMDV infection-induced autophagy plays an important role in viral replication. ( A ) PK-15 cells were mock infected or infected with FMDV (MOI = 1) for 0, 0.5, 1, 1.5, 2 and 3 h. The expression of LC3B and SQSTM1 was analyzed by western blot. The level of 3D was analyzed by RT-PCR. ACTB and GAPDH were used as a sample loading control. ( B ) The cells were then fixed and processed for indirect immunofluorescence using antibodies against LC3B and the 3D protein, followed by the corresponding secondary antibodies. The fluorescence signals were visualized by confocal immunofluorescence microscopy. ( C ) ATG5 knockdown (KD) and wild-type cells were infected with FMDV (MOI = 1) for 2 and 3 h. The expression of ATG5 and LC3B was analyzed by western blot. ( D and E ) ATG5 KD and wild-type cells were infected with FMDV (MOI = 1). At 3 hpi, both the extracellular and intracellular copy numbers of FMDV were measured by qRT-PCR; both the extracellular and intracellular virus titers were measured by TCID 50 . The data represent the mean ± SD of 3 independent experiments. ***P < 0.001. ( F ) PK-15 cells were mock infected or infected with FMDV (MOI = 1) for 6 and 9 h. The expression of SQSTM1 and VP1 were analyzed by western blot. ( G ) Cells were transfected with pmCherry-GFP-LC3B for 24 h, followed by FMDV infection (MOI = 1) and treatment with Baf-A1. The fluorescence signals were visualized by confocal immunofluorescence microscopy. Scale bars: 10 μm.

    Techniques Used: Infection, Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Fluorescence, Microscopy, Quantitative RT-PCR, Transfection

    FMDV infection induces autophagy through the EIF2S1-ATF4-AKT-MTOR cascade. ( A ) PK-15 cells were mock infected or infected with FMDV (MOI = 1) for 1.5, 2 and 3 h. The phosphorylation of AKT, AMPK, MTOR and ULK1 were analyzed by western blot. ACTB was used as a sample loading control. ( B ) PK-15 cells were mock infected or infected with FMDV (MOI = 1) for 1.5, 2 and 3 h. ATF4 and phosphorylation of EIF2S1 were analyzed by western blot. ( C ) ATF4 KD and wild-type cells cells were infected with FMDV (MOI = 1). ATF4, LC3B and phosphorylation of AKT, MTOR and ULK1 were analyzed by western blot. ( D ) ATF4 and scrambled knockdown cells were transfected with pmCherry-GFP-LC3B for 24 h, followed by FMDV infection (MOI = 1) for 3 h. The fluorescence signals were visualized by confocal immunofluorescence microscopy. Scale bars: 10 μm. ( E and F ) ATF4 KD and wil-type cells cells were infected with FMDV (MOI = 1) for 3 h. At 3 hpi, both the extracellular and intracellular copy numbers of FMDV were measured by qRT-PCR; both the extracellular and intracellular virus titers were measured by TCID 50 . The data represent the mean ± SD of 3 independent experiments. **P < 0.01, ***P < 0.001.
    Figure Legend Snippet: FMDV infection induces autophagy through the EIF2S1-ATF4-AKT-MTOR cascade. ( A ) PK-15 cells were mock infected or infected with FMDV (MOI = 1) for 1.5, 2 and 3 h. The phosphorylation of AKT, AMPK, MTOR and ULK1 were analyzed by western blot. ACTB was used as a sample loading control. ( B ) PK-15 cells were mock infected or infected with FMDV (MOI = 1) for 1.5, 2 and 3 h. ATF4 and phosphorylation of EIF2S1 were analyzed by western blot. ( C ) ATF4 KD and wild-type cells cells were infected with FMDV (MOI = 1). ATF4, LC3B and phosphorylation of AKT, MTOR and ULK1 were analyzed by western blot. ( D ) ATF4 and scrambled knockdown cells were transfected with pmCherry-GFP-LC3B for 24 h, followed by FMDV infection (MOI = 1) for 3 h. The fluorescence signals were visualized by confocal immunofluorescence microscopy. Scale bars: 10 μm. ( E and F ) ATF4 KD and wil-type cells cells were infected with FMDV (MOI = 1) for 3 h. At 3 hpi, both the extracellular and intracellular copy numbers of FMDV were measured by qRT-PCR; both the extracellular and intracellular virus titers were measured by TCID 50 . The data represent the mean ± SD of 3 independent experiments. **P < 0.01, ***P < 0.001.

    Techniques Used: Infection, Western Blot, Transfection, Fluorescence, Immunofluorescence, Microscopy, Quantitative RT-PCR

    UV-FMDV infection induces autophagy. ( A ) PK-15 cells were mock infected or infected with UV-FMDV for 3 h (MOI = 10). LC3B and phosphorylation of EIF2S1, AKT and MTOR were analyzed by western blot. ACTB was used as a sample loading control. ( B ) ATF4 KD and wild-type cells were infected as described in ( A ).
    Figure Legend Snippet: UV-FMDV infection induces autophagy. ( A ) PK-15 cells were mock infected or infected with UV-FMDV for 3 h (MOI = 10). LC3B and phosphorylation of EIF2S1, AKT and MTOR were analyzed by western blot. ACTB was used as a sample loading control. ( B ) ATF4 KD and wild-type cells were infected as described in ( A ).

    Techniques Used: Infection, Western Blot

    FMDV capsid protein VP2 induced autophagy. ( A ) PK-15 cells were transfected with empty vectors or various plasmids expressing FLAG-tagged VP1, VP2and VP3 proteins for 24 h. LC3B and FMDV capsid proteins were analyzed by western blot. ACTB was used as a sample loading control. ( B ) Cells were transfected with empty vectors or pCMV-Flag-VP2 for 24 h. Cells were fixed and analyzed by immunofluorescence using anti-LC3B antibodies. The fluorescence signals were visualized by confocal immunofluorescence microscopy. Scale bars: 10 μm. ( C ) PK-15 cells were transfected with empty vectors or pCMV-Flag-VP2 for 24 h. LC3B and phosphorylation of EIF2S1, AKT and MTOR were analyzed by western blot. ACTB was used as a sample loading control. ( D ) ATF4 and scrambled knockdown cells were transfected with pCMV-Flag-VP2 for 24 h. LC3B and phosphorylation of AKT and MTOR were analyzed by western blot. ACTB was used as a sample loading control.
    Figure Legend Snippet: FMDV capsid protein VP2 induced autophagy. ( A ) PK-15 cells were transfected with empty vectors or various plasmids expressing FLAG-tagged VP1, VP2and VP3 proteins for 24 h. LC3B and FMDV capsid proteins were analyzed by western blot. ACTB was used as a sample loading control. ( B ) Cells were transfected with empty vectors or pCMV-Flag-VP2 for 24 h. Cells were fixed and analyzed by immunofluorescence using anti-LC3B antibodies. The fluorescence signals were visualized by confocal immunofluorescence microscopy. Scale bars: 10 μm. ( C ) PK-15 cells were transfected with empty vectors or pCMV-Flag-VP2 for 24 h. LC3B and phosphorylation of EIF2S1, AKT and MTOR were analyzed by western blot. ACTB was used as a sample loading control. ( D ) ATF4 and scrambled knockdown cells were transfected with pCMV-Flag-VP2 for 24 h. LC3B and phosphorylation of AKT and MTOR were analyzed by western blot. ACTB was used as a sample loading control.

    Techniques Used: Transfection, Expressing, Western Blot, Immunofluorescence, Fluorescence, Microscopy

    VP2 mutant and interaction between VP2 and HSPB1 in PK-15 cells. ( A ) A scheme of the VP2 muant. ( B ) PK-15 cells were transfected with empty vectors, pCMV-Flag-VP2, or VP2 mutant for 24 h. LC3B and ACTB were analyzed by western blot. ( C and D ) The interaction between FMDV VP2 and HSPB1 in PK-15 cells was verified. The PK-15 cells were co-transfected with 10 μg pCMV-Flag-VP2 plasmid and 10 μg pEGFP-HA-HSPB1 plasmid or transfected with just 10 μg pCMV-Flag-VP2 plasmid, and immunoprecipitation was performed with anti-HA antibody. Immunoblotting analysis was performed with anti-HA antibody and anti-FLAG antibody. ( E and F ) The PK-15 cells were transfected with 10 μg pCMV-Flag-VP2 plasmid or 10 μg empty vector pCMV-Flag plasmid, and immunoprecipitation was performed with anti-FLAG antibody. Immunoblotting analysis was performed with anti-FLAG antibody and anti-HSPB1 antibody.
    Figure Legend Snippet: VP2 mutant and interaction between VP2 and HSPB1 in PK-15 cells. ( A ) A scheme of the VP2 muant. ( B ) PK-15 cells were transfected with empty vectors, pCMV-Flag-VP2, or VP2 mutant for 24 h. LC3B and ACTB were analyzed by western blot. ( C and D ) The interaction between FMDV VP2 and HSPB1 in PK-15 cells was verified. The PK-15 cells were co-transfected with 10 μg pCMV-Flag-VP2 plasmid and 10 μg pEGFP-HA-HSPB1 plasmid or transfected with just 10 μg pCMV-Flag-VP2 plasmid, and immunoprecipitation was performed with anti-HA antibody. Immunoblotting analysis was performed with anti-HA antibody and anti-FLAG antibody. ( E and F ) The PK-15 cells were transfected with 10 μg pCMV-Flag-VP2 plasmid or 10 μg empty vector pCMV-Flag plasmid, and immunoprecipitation was performed with anti-FLAG antibody. Immunoblotting analysis was performed with anti-FLAG antibody and anti-HSPB1 antibody.

    Techniques Used: Mutagenesis, Transfection, Western Blot, Plasmid Preparation, Immunoprecipitation

    VP2 decreases aggregation of HTT103Q. ( A ) HEK293T cells and ( B) Vero cells were transfected with empty vectors or pCMV-N-VP2 for 24 h. LC3B and SQSTM1 by were analyzed by western blot. ACTB was used as a sample loading control. ( C ) PK-15 cells were transfected with empty vectors or pCMV-EGFP-HTT103Q for 24 h. HTT103Q and SQSTM1 were analyzed by western blot. ACTB was used as a sample loading control. ( D ) PK-15 cells and ATG5 KD cells were co-transfected with pCMV-Flag-VP2 and pCMV-EGFP-HTT103Q for 24 h. The fluorescence signals were visualized by confocal immunofluorescence microscopy. Scale bars: 10 μm. ( E ) The number of HTT103Q dot was counted. The data represent the mean ± SD of 3 independent experiments. **P < 0.01.
    Figure Legend Snippet: VP2 decreases aggregation of HTT103Q. ( A ) HEK293T cells and ( B) Vero cells were transfected with empty vectors or pCMV-N-VP2 for 24 h. LC3B and SQSTM1 by were analyzed by western blot. ACTB was used as a sample loading control. ( C ) PK-15 cells were transfected with empty vectors or pCMV-EGFP-HTT103Q for 24 h. HTT103Q and SQSTM1 were analyzed by western blot. ACTB was used as a sample loading control. ( D ) PK-15 cells and ATG5 KD cells were co-transfected with pCMV-Flag-VP2 and pCMV-EGFP-HTT103Q for 24 h. The fluorescence signals were visualized by confocal immunofluorescence microscopy. Scale bars: 10 μm. ( E ) The number of HTT103Q dot was counted. The data represent the mean ± SD of 3 independent experiments. **P < 0.01.

    Techniques Used: Transfection, Western Blot, Fluorescence, Immunofluorescence, Microscopy

    swine kidney cell line pk 15  (ATCC)


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    ATCC swine kidney cell line pk 15
    Swine Kidney Cell Line Pk 15, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    swine kidney cell line pk 15  (ATCC)


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    ATCC swine kidney cell line pk 15
    CSFV infection upregulated PKM2 expression. ( A ) Immunohistochemical analysis of PKM2 expression in normal and CSFV-infected tissues. ( B ) RT-qPCR analysis of PKM2 gene expression in normal and CSFV-infected tissues. Error bars indicate the mean (±SD) of three independent experiments. * P < 0.05; *** P < 0.001; **** P < 0.0001, and ns, P > 0.05 (one-way ANOVA). ( C ) RT-qPCR analysis of PKM2 gene transcription in CSFV-infected <t>PK-15</t> and 3D4/2 cells. Error bars indicate the mean (±SD) of three independent experiments. n = 3. * P < 0.05; ** P < 0.01; ***P < 0.001; and ns, P > 0.05 (one-way ANOVA). ( D ) Western blot analysis of PKM2 protein expression in CSFV-infected PK-15 and 3D4/2 cells. The level of protein was quantified using Image-Pro Plus 6.0 software, and the ratios were calculated relative to the tubulin control. Error bars indicate the mean (±SD) of three independent experiments. **** P < 0.0001 (one-way ANOVA).
    Swine Kidney Cell Line Pk 15, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/swine kidney cell line pk 15/product/ATCC
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    swine kidney cell line pk 15 - by Bioz Stars, 2024-06
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    1) Product Images from "PKM2 induces mitophagy through the AMPK-mTOR pathway promoting CSFV proliferation"

    Article Title: PKM2 induces mitophagy through the AMPK-mTOR pathway promoting CSFV proliferation

    Journal: Journal of Virology

    doi: 10.1128/jvi.01751-23

    CSFV infection upregulated PKM2 expression. ( A ) Immunohistochemical analysis of PKM2 expression in normal and CSFV-infected tissues. ( B ) RT-qPCR analysis of PKM2 gene expression in normal and CSFV-infected tissues. Error bars indicate the mean (±SD) of three independent experiments. * P < 0.05; *** P < 0.001; **** P < 0.0001, and ns, P > 0.05 (one-way ANOVA). ( C ) RT-qPCR analysis of PKM2 gene transcription in CSFV-infected PK-15 and 3D4/2 cells. Error bars indicate the mean (±SD) of three independent experiments. n = 3. * P < 0.05; ** P < 0.01; ***P < 0.001; and ns, P > 0.05 (one-way ANOVA). ( D ) Western blot analysis of PKM2 protein expression in CSFV-infected PK-15 and 3D4/2 cells. The level of protein was quantified using Image-Pro Plus 6.0 software, and the ratios were calculated relative to the tubulin control. Error bars indicate the mean (±SD) of three independent experiments. **** P < 0.0001 (one-way ANOVA).
    Figure Legend Snippet: CSFV infection upregulated PKM2 expression. ( A ) Immunohistochemical analysis of PKM2 expression in normal and CSFV-infected tissues. ( B ) RT-qPCR analysis of PKM2 gene expression in normal and CSFV-infected tissues. Error bars indicate the mean (±SD) of three independent experiments. * P < 0.05; *** P < 0.001; **** P < 0.0001, and ns, P > 0.05 (one-way ANOVA). ( C ) RT-qPCR analysis of PKM2 gene transcription in CSFV-infected PK-15 and 3D4/2 cells. Error bars indicate the mean (±SD) of three independent experiments. n = 3. * P < 0.05; ** P < 0.01; ***P < 0.001; and ns, P > 0.05 (one-way ANOVA). ( D ) Western blot analysis of PKM2 protein expression in CSFV-infected PK-15 and 3D4/2 cells. The level of protein was quantified using Image-Pro Plus 6.0 software, and the ratios were calculated relative to the tubulin control. Error bars indicate the mean (±SD) of three independent experiments. **** P < 0.0001 (one-way ANOVA).

    Techniques Used: Infection, Expressing, Immunohistochemical staining, Quantitative RT-PCR, Western Blot, Software

    CSFV affects pyruvate metabolism in PK-15 and 34D/2 cells through PKM2. ( A and B ) PK-15 ( A ) and 3D4/2 cells ( B ) were infected with CSFV at an MOI of 1.0 and pyruvate content was measured by spectrophotometric assays in cultured cells at 24 and 48 h. ( C ) PK-15 cells were transfected with PKM2 and then infected with CSFV at an MOI of 1.0. The content of pyruvate in cultured cells was measured spectrophotometrically at 24 and 48 h. ( D ) 3D4/2 cells were transfected with PKM2 and then infected with CSFV at MOI of 1.0. The content of pyruvate in cultured cells was measured spectrophotometrically at 24 and 48 h. All measurements were made in triplicates. Error bars indicate the mean (±SD) of three independent experiments. * P < 0.05; ** P < 0.01; and *** P < 0.001 (one-way ANOVA).
    Figure Legend Snippet: CSFV affects pyruvate metabolism in PK-15 and 34D/2 cells through PKM2. ( A and B ) PK-15 ( A ) and 3D4/2 cells ( B ) were infected with CSFV at an MOI of 1.0 and pyruvate content was measured by spectrophotometric assays in cultured cells at 24 and 48 h. ( C ) PK-15 cells were transfected with PKM2 and then infected with CSFV at an MOI of 1.0. The content of pyruvate in cultured cells was measured spectrophotometrically at 24 and 48 h. ( D ) 3D4/2 cells were transfected with PKM2 and then infected with CSFV at MOI of 1.0. The content of pyruvate in cultured cells was measured spectrophotometrically at 24 and 48 h. All measurements were made in triplicates. Error bars indicate the mean (±SD) of three independent experiments. * P < 0.05; ** P < 0.01; and *** P < 0.001 (one-way ANOVA).

    Techniques Used: Infection, Cell Culture, Transfection

    Validation of the interaction of PKM2 with NS4A and NS5A by mass spectrometry and immunoprecipitation. ( A ) Mass spectrometry screening of host-protein networks interacting with CSFV NS4A. ( B ) Analysis of the CSFV NS4A protein-host protein interaction network. ( C ) Statistical analysis of host proteins related to metabolism based on MCC using the cytoHubba plugin. ( D ) Statistical analysis of host proteins related to metabolism by setting continuous gradients based on degree values. ( E ) Co-immunoprecipitation analysis of 3×Flag-tagged PKM2 and GFP-tagged NS4A and NS5A by the anti-flag monoclonal antibody (mAb) or by the anti-GFP mAb. PK-15 and 3D4/2 cells were co-transfected with the indicated plasmids (+) or empty vectors (−) for 24 h. The transfected cells were lysed and incubated with a mouse anti-Flag mAb or anti-GFP mAb, followed by incubation with the protein G-agarose for 6 h at 4°C. The immunoprecipitate was analyzed by western blot using the anti-Flag and anti-GFP.
    Figure Legend Snippet: Validation of the interaction of PKM2 with NS4A and NS5A by mass spectrometry and immunoprecipitation. ( A ) Mass spectrometry screening of host-protein networks interacting with CSFV NS4A. ( B ) Analysis of the CSFV NS4A protein-host protein interaction network. ( C ) Statistical analysis of host proteins related to metabolism based on MCC using the cytoHubba plugin. ( D ) Statistical analysis of host proteins related to metabolism by setting continuous gradients based on degree values. ( E ) Co-immunoprecipitation analysis of 3×Flag-tagged PKM2 and GFP-tagged NS4A and NS5A by the anti-flag monoclonal antibody (mAb) or by the anti-GFP mAb. PK-15 and 3D4/2 cells were co-transfected with the indicated plasmids (+) or empty vectors (−) for 24 h. The transfected cells were lysed and incubated with a mouse anti-Flag mAb or anti-GFP mAb, followed by incubation with the protein G-agarose for 6 h at 4°C. The immunoprecipitate was analyzed by western blot using the anti-Flag and anti-GFP.

    Techniques Used: Mass Spectrometry, Immunoprecipitation, Transfection, Incubation, Western Blot

    PKM2 colocalized with CSFV NS4A and NS5A and promoted the expression of NS4A and NS5A ( A and B ). PK-15 ( A ) and 3D4/2 ( B ) cells were co-transfected with 3× Flag-tagged PKM2 and GFP-tagged NS4A/NS5A. Cells were fixed at 24 h post-transfection and subjected to indirect immunofluorescence assay to detect GFP-NS4A/NS5A (green) and 3× Flag-PKM2 (red) with mouse anti-Flag and rabbit anti-GFP antibodies. The merged image indicates the nucleus by 4′,6-diamidino-2-phenylindole (DAPI) (blue) staining. ( C ) Western blot detection of NS4A protein expression levels in PK-15 cells overexpressing or silencing PKM2. ( D ) Western blot detection of NS5A protein expression levels in PK-15 cells overexpressing or silencing PKM2.
    Figure Legend Snippet: PKM2 colocalized with CSFV NS4A and NS5A and promoted the expression of NS4A and NS5A ( A and B ). PK-15 ( A ) and 3D4/2 ( B ) cells were co-transfected with 3× Flag-tagged PKM2 and GFP-tagged NS4A/NS5A. Cells were fixed at 24 h post-transfection and subjected to indirect immunofluorescence assay to detect GFP-NS4A/NS5A (green) and 3× Flag-PKM2 (red) with mouse anti-Flag and rabbit anti-GFP antibodies. The merged image indicates the nucleus by 4′,6-diamidino-2-phenylindole (DAPI) (blue) staining. ( C ) Western blot detection of NS4A protein expression levels in PK-15 cells overexpressing or silencing PKM2. ( D ) Western blot detection of NS5A protein expression levels in PK-15 cells overexpressing or silencing PKM2.

    Techniques Used: Expressing, Transfection, Immunofluorescence, Staining, Western Blot

    PKM2 positively regulated the proliferation of CSFV in PK-15 and 3D4/2 cells ( A and D ). PK-15 and 3D4/2 cells were transduced with p3×Flag-PKM2 or p3×Flag-CMV ( A ) (siNC or siPKM2) ( D ), followed by infection with CSFV at an MOI of 1.0 or mock infected. Cell samples were analyzed by western blot with antibodies against PKM2, CSFV E2, and tubulin (loading control). ( B and E ) CSFV virus titers in the supernatant were determined as 50% tissue culture infective doses (TCID50)/mL as described in Materials and Methods. Error bars indicate the mean (±SD) of three independent experiments. * P < 0.05; ** P < 0.01; and *** P < 0.001 (one-way ANOVA). ( C ) siRNA knockdown of PKM2 in PK-15 and 3D4/2 cells transfected with siNC or PKM2 siRNA-1/-2/-3. The expression of PKM2 was assessed by western blot and RT-qPCR at 24 h. Error bars indicate the mean (±SD) of three independent experiments. *** P < 0.001 and **** P < 0.0001 (one-way ANOVA).
    Figure Legend Snippet: PKM2 positively regulated the proliferation of CSFV in PK-15 and 3D4/2 cells ( A and D ). PK-15 and 3D4/2 cells were transduced with p3×Flag-PKM2 or p3×Flag-CMV ( A ) (siNC or siPKM2) ( D ), followed by infection with CSFV at an MOI of 1.0 or mock infected. Cell samples were analyzed by western blot with antibodies against PKM2, CSFV E2, and tubulin (loading control). ( B and E ) CSFV virus titers in the supernatant were determined as 50% tissue culture infective doses (TCID50)/mL as described in Materials and Methods. Error bars indicate the mean (±SD) of three independent experiments. * P < 0.05; ** P < 0.01; and *** P < 0.001 (one-way ANOVA). ( C ) siRNA knockdown of PKM2 in PK-15 and 3D4/2 cells transfected with siNC or PKM2 siRNA-1/-2/-3. The expression of PKM2 was assessed by western blot and RT-qPCR at 24 h. Error bars indicate the mean (±SD) of three independent experiments. *** P < 0.001 and **** P < 0.0001 (one-way ANOVA).

    Techniques Used: Transduction, Infection, Western Blot, Virus, Transfection, Expressing, Quantitative RT-PCR

    Rescue effect of pyruvate on silencing PKM2-induced inhibition of CSFV replication. ( A ) PK-15 and 3D4/2 cells were cultured in a pyruvate-free medium for a while after the addition of pyruvate solution, respectively, followed by infection with CSFV at an MOI of 1.0, and CSFV N pro protein expression was detected after 48 h. ( B ) The relative expression level of NS5B gene in PK-15 and 3D4/2 cells was analyzed by RT-qPCR. Cells were treated as in panel A . ( C ) PK-15 and 3D4/2 cells were treated with pyruvate (5 mM) after being transfected with siPKM2 and then infected with CSFV at an MOI of 1.0. At 48 hpi, cell samples were analyzed by western blot with antibodies against CSFV E2 and tubulin (loading control). ( D ) PK-15 and 3D4/2 cells were treated as in panel C . NS5B gene levels in PK-15 and 3D4/2 cells were assessed using RT-qPCR. Error bars indicate the mean (±SD) of three independent experiments. ns, P > 0.05; ** P < 0.01; and *** P < 0.001 (one-way ANOVA). ( E ) PK-15 and 3D4/2 cells were treated as in panel C . CSFV virus titers in the supernatant were determined as 50% tissue culture infective doses (TCID50)/mL as described in Materials and Methods. Error bars indicate the mean (±SD) of three independent experiments. ns, P > 0.05 and ** P < 0.01 (one-way ANOVA).
    Figure Legend Snippet: Rescue effect of pyruvate on silencing PKM2-induced inhibition of CSFV replication. ( A ) PK-15 and 3D4/2 cells were cultured in a pyruvate-free medium for a while after the addition of pyruvate solution, respectively, followed by infection with CSFV at an MOI of 1.0, and CSFV N pro protein expression was detected after 48 h. ( B ) The relative expression level of NS5B gene in PK-15 and 3D4/2 cells was analyzed by RT-qPCR. Cells were treated as in panel A . ( C ) PK-15 and 3D4/2 cells were treated with pyruvate (5 mM) after being transfected with siPKM2 and then infected with CSFV at an MOI of 1.0. At 48 hpi, cell samples were analyzed by western blot with antibodies against CSFV E2 and tubulin (loading control). ( D ) PK-15 and 3D4/2 cells were treated as in panel C . NS5B gene levels in PK-15 and 3D4/2 cells were assessed using RT-qPCR. Error bars indicate the mean (±SD) of three independent experiments. ns, P > 0.05; ** P < 0.01; and *** P < 0.001 (one-way ANOVA). ( E ) PK-15 and 3D4/2 cells were treated as in panel C . CSFV virus titers in the supernatant were determined as 50% tissue culture infective doses (TCID50)/mL as described in Materials and Methods. Error bars indicate the mean (±SD) of three independent experiments. ns, P > 0.05 and ** P < 0.01 (one-way ANOVA).

    Techniques Used: Inhibition, Cell Culture, Infection, Expressing, Quantitative RT-PCR, Transfection, Western Blot, Virus

    PKM2 increased ROS in cells and disrupted mitochondrial function. ( A ) PKM2 overexpression and CSFV infection increased ROS in PK-15 cells. Rosup as a positive control. ( B and C ) The Mitochondrial Membrane Potential Assay Kit (JC-1) detected mitochondrial membrane potential in PK-15 ( B ) and 3D4/2 cells ( C ). In normal mitochondria, JC-1 is present as a polymer with bright red fluorescence and very weak green fluorescence in the cell. When the mitochondrial membrane potential is reduced by treatment with carbonyl cyanide m-chlorobenzyl hydrazone (CCCP), JC-1 cannot be present as a polymer in the mitochondrial matrix, and the intensity of red fluorescence in the mitochondria is significantly reduced. In contrast, green fluorescence in the cytoplasm is enhanced considerably. Image-Pro Plus 6.0 software was used to calculate the mean fluorescence intensity of the line profile of the merged image (three times).
    Figure Legend Snippet: PKM2 increased ROS in cells and disrupted mitochondrial function. ( A ) PKM2 overexpression and CSFV infection increased ROS in PK-15 cells. Rosup as a positive control. ( B and C ) The Mitochondrial Membrane Potential Assay Kit (JC-1) detected mitochondrial membrane potential in PK-15 ( B ) and 3D4/2 cells ( C ). In normal mitochondria, JC-1 is present as a polymer with bright red fluorescence and very weak green fluorescence in the cell. When the mitochondrial membrane potential is reduced by treatment with carbonyl cyanide m-chlorobenzyl hydrazone (CCCP), JC-1 cannot be present as a polymer in the mitochondrial matrix, and the intensity of red fluorescence in the mitochondria is significantly reduced. In contrast, green fluorescence in the cytoplasm is enhanced considerably. Image-Pro Plus 6.0 software was used to calculate the mean fluorescence intensity of the line profile of the merged image (three times).

    Techniques Used: Over Expression, Infection, Positive Control, Membrane, Polymer, Fluorescence, Software

    PKM2 induced mitochondrial fission and mitophagy. ( A ) Confocal microscopy images showing mitochondrial fragmentation in PKM2-overexpressed cells. PK-15 and 3D4/2 cells were transfected with P3×Flag-CMV or Flag-PKM2 for 24 h. Cells have stained the mitochondria with MitoTracker (red) and the cell nuclei with 4′,6-diamidino-2-phenylindole (DAPI) (blue). In the zoomed images, typical tubular mitochondria in control cells and fragmented mitochondria in PKM2-overexpressed cells are shown. The bar graph represents the average number of mitochondria (red dots) in each cell. Results represent the mean of at least three independent experiments. ** P < 0.01; *** P < 0.001, and **** P < 0.0001. ( B ) Confocal microscope image showing co-localization of PKM2 with TOM20. Cells were prepared as in panel A . At 24 h, cells were immunostained with the TOM20 antibody (red) and 4′,6-diamidino-2-phenylindole (DAPI) (blue). Image-Pro Plus6.0 software was used to calculate the mean fluorescence intensity of the line profile of the merged image (three times). ** P < 0.01 and *** P < 0.001 (one-way ANOVA). ( C and D ) Western blot detected the relative expression of the autophagy-associated proteins ATG5, LC3, and p62 in cells with PKM2 overexpression ( C ) and inhibition ( D ). The marker proteins TOM20, VDACI, COXIV, LAMP1, and tubulin (loading control) of mitophagy were also detected by western blot. The level of protein and fluorescence intensity were quantified using Image-Pro Plus 6.0 software. Error bars indicate the mean (±SD) of three independent experiments. ** P < 0.01; *** P < 0.001, and **** P < 0.0001 (one-way ANOVA).
    Figure Legend Snippet: PKM2 induced mitochondrial fission and mitophagy. ( A ) Confocal microscopy images showing mitochondrial fragmentation in PKM2-overexpressed cells. PK-15 and 3D4/2 cells were transfected with P3×Flag-CMV or Flag-PKM2 for 24 h. Cells have stained the mitochondria with MitoTracker (red) and the cell nuclei with 4′,6-diamidino-2-phenylindole (DAPI) (blue). In the zoomed images, typical tubular mitochondria in control cells and fragmented mitochondria in PKM2-overexpressed cells are shown. The bar graph represents the average number of mitochondria (red dots) in each cell. Results represent the mean of at least three independent experiments. ** P < 0.01; *** P < 0.001, and **** P < 0.0001. ( B ) Confocal microscope image showing co-localization of PKM2 with TOM20. Cells were prepared as in panel A . At 24 h, cells were immunostained with the TOM20 antibody (red) and 4′,6-diamidino-2-phenylindole (DAPI) (blue). Image-Pro Plus6.0 software was used to calculate the mean fluorescence intensity of the line profile of the merged image (three times). ** P < 0.01 and *** P < 0.001 (one-way ANOVA). ( C and D ) Western blot detected the relative expression of the autophagy-associated proteins ATG5, LC3, and p62 in cells with PKM2 overexpression ( C ) and inhibition ( D ). The marker proteins TOM20, VDACI, COXIV, LAMP1, and tubulin (loading control) of mitophagy were also detected by western blot. The level of protein and fluorescence intensity were quantified using Image-Pro Plus 6.0 software. Error bars indicate the mean (±SD) of three independent experiments. ** P < 0.01; *** P < 0.001, and **** P < 0.0001 (one-way ANOVA).

    Techniques Used: Confocal Microscopy, Transfection, Staining, Microscopy, Software, Fluorescence, Western Blot, Expressing, Over Expression, Inhibition, Marker

    PKM2 induced complete mitophagy. ( A and B ) Overexpression of PKM2 increased mitochondrial autophagic vesicle formation in PK-15 ( A ) and 3D4/2 cells ( B ). TEM images revealed the mitochondrial ultrastructure in PKM2-overexpressed cells. PK-15 and 3D4/2 cells were mock handled or PKM2 overexpressed for 24 h and analyzed by TEM. Typical elongated tubular mitochondria in mock cells and fragmented elliptic mitochondria engulfed with membrane-like vesicles in PKM2-overexpressed cells were observed in the zoomed images. Scale bar: 2 µm. ( C ) Quantification of the mitophagosome-like vesicles per cell image (mean ± SD; n ≥ 5 cells; **** P < 0.0001) (one-way ANOVA). ( D and E ) PK-15 ( D ) and 3D4/2 cells ( E ) transiently expressing Mito-mRFP-EGFP were transfected with P3×Flag-CMV or Flag-PKM2 for 24 h. In the zoomed images, fluorescence signals indicated the expression of mRFP and GFP protein targeting mitochondria: yellow color, no mitophagy; red color, mitophagy. Image-Pro Plus 6.0 software was used to measure the fluorescence intensity quantitatively. Bar graphs represent the mean number of autophagosomes (puncta with both red and green colors, i.e., puncta with yellow color in merged images) and autolysosomes (puncta with only red but not green color, i.e., puncta with red color in merged images) per cell. Error bars indicate the mean (±SD) of three independent experiments. ns, P > 0.05 and **** P < 0.0001 (two-way ANOVA).
    Figure Legend Snippet: PKM2 induced complete mitophagy. ( A and B ) Overexpression of PKM2 increased mitochondrial autophagic vesicle formation in PK-15 ( A ) and 3D4/2 cells ( B ). TEM images revealed the mitochondrial ultrastructure in PKM2-overexpressed cells. PK-15 and 3D4/2 cells were mock handled or PKM2 overexpressed for 24 h and analyzed by TEM. Typical elongated tubular mitochondria in mock cells and fragmented elliptic mitochondria engulfed with membrane-like vesicles in PKM2-overexpressed cells were observed in the zoomed images. Scale bar: 2 µm. ( C ) Quantification of the mitophagosome-like vesicles per cell image (mean ± SD; n ≥ 5 cells; **** P < 0.0001) (one-way ANOVA). ( D and E ) PK-15 ( D ) and 3D4/2 cells ( E ) transiently expressing Mito-mRFP-EGFP were transfected with P3×Flag-CMV or Flag-PKM2 for 24 h. In the zoomed images, fluorescence signals indicated the expression of mRFP and GFP protein targeting mitochondria: yellow color, no mitophagy; red color, mitophagy. Image-Pro Plus 6.0 software was used to measure the fluorescence intensity quantitatively. Bar graphs represent the mean number of autophagosomes (puncta with both red and green colors, i.e., puncta with yellow color in merged images) and autolysosomes (puncta with only red but not green color, i.e., puncta with red color in merged images) per cell. Error bars indicate the mean (±SD) of three independent experiments. ns, P > 0.05 and **** P < 0.0001 (two-way ANOVA).

    Techniques Used: Over Expression, Membrane, Expressing, Transfection, Fluorescence, Software

    Silencing PKM2 inhibited mitophagy induced by CSFV. ( A and B ) PK-15 ( A ) and 3D4/2 cells ( B ) were transfected with the siRNA of siPKM2 or siNC for 24 h, then mock infected or infected with CSFV (MOI = 1.0). Western blot was used to analyze the relative expression of proteins ATG5, P62, LC3, COXIV, TOM20, and tubulin (loading control). The level of protein was quantified using Image-Pro Plus 6.0 software. Error bars indicate the mean (±SD) of three independent experiments. * P < 0.05; ** P < 0.01, and *** P < 0.001 (one-way ANOVA). ( C and D ) PK-15 and 3D4/2 cells transiently expressing Mito-mRFP-EGFP were transfected with siPKM2 or infected with CSFV for 24 h. In the zoomed images, fluorescence signals indicated the expression of mRFP and GFP proteins targeting mitochondria: yellow color, no mitophagy; red color, mitophagy. Image-Pro Plus 6.0 software was used to measure the fluorescence intensity quantitatively. Bar graphs represent the mean number of autophagosomes (puncta with both red and green colors, i.e., puncta with yellow color in merged images) and autolysosomes (puncta with only red but not green color, i.e., puncta with red color in merged images) per cell. Error bars indicate the mean (±SD) of three independent experiments. ns, P > 0.05 and **** P < 0.0001 (two-way ANOVA).
    Figure Legend Snippet: Silencing PKM2 inhibited mitophagy induced by CSFV. ( A and B ) PK-15 ( A ) and 3D4/2 cells ( B ) were transfected with the siRNA of siPKM2 or siNC for 24 h, then mock infected or infected with CSFV (MOI = 1.0). Western blot was used to analyze the relative expression of proteins ATG5, P62, LC3, COXIV, TOM20, and tubulin (loading control). The level of protein was quantified using Image-Pro Plus 6.0 software. Error bars indicate the mean (±SD) of three independent experiments. * P < 0.05; ** P < 0.01, and *** P < 0.001 (one-way ANOVA). ( C and D ) PK-15 and 3D4/2 cells transiently expressing Mito-mRFP-EGFP were transfected with siPKM2 or infected with CSFV for 24 h. In the zoomed images, fluorescence signals indicated the expression of mRFP and GFP proteins targeting mitochondria: yellow color, no mitophagy; red color, mitophagy. Image-Pro Plus 6.0 software was used to measure the fluorescence intensity quantitatively. Bar graphs represent the mean number of autophagosomes (puncta with both red and green colors, i.e., puncta with yellow color in merged images) and autolysosomes (puncta with only red but not green color, i.e., puncta with red color in merged images) per cell. Error bars indicate the mean (±SD) of three independent experiments. ns, P > 0.05 and **** P < 0.0001 (two-way ANOVA).

    Techniques Used: Transfection, Infection, Western Blot, Expressing, Software, Fluorescence

    The AMPK-mTOR pathway was upregulated in PKM2-inhibited cells with CSFV infection. ( A and B ) PK-15 and 3D4/2 cells were transfected with p3×Flag-CMV or Flag-PKM2 and ( A ) siPKM2 or siNC ( B ). Cell samples were analyzed at 24 and 48 h by immunoblotting with antibodies against AMPK, p-AMPK, p-mTOR, Flag, PKM2, and tubulin (loading control). ( C and D ) PK-15 and 3D4/2 cells were treated with 10 µM Compound C ( C ) or 10 µM AICAR ( D ). DMSO treatment was used as a control. Cell samples were analyzed at 24 h by immunoblotting with antibodies against AMPK and tubulin (loading control). Bar graphs represent the mean number of autophagosomes (puncta with both red and green colors, i.e., puncta with yellow color in merged images) and autolysosomes (puncta with only red but not green color, i.e., puncta with red color in merged images) per cell. Error bars indicate the mean (±SD) of three independent experiments. ns, P > 0.05; *** P < 0.001, and **** P < 0.0001 (two-way ANOVA). ( E ) PK-15 and 3D4/2 cells were treated with DMSO or Compound C and then transfected with p3×Flag-CMV or Flag-PKM2. Cell samples were analyzed at 24 h by immunoblotting with antibodies against Flag, p-mTOR, and tubulin (loading control). ( F ) PK-15 and 3D4/2 cells were treated with 10 µM DMSO or 10 µM AICAR and then transfected with siPKM2 or siNC. Cell samples were analyzed at 24 h by immunoblotting with antibodies against PKM2, p-mTOR, and tubulin (loading control). ( G ) PK-15 and 3D4/2 cells were transfected with the siPKM2 or siNC for 24 h and then mock infected or infected with CSFV (MOI = 1.0). Western blot was used to analyze the relative expression of proteins AMPK, p-AMPK, p-mTOR, and TUBA (loading control).
    Figure Legend Snippet: The AMPK-mTOR pathway was upregulated in PKM2-inhibited cells with CSFV infection. ( A and B ) PK-15 and 3D4/2 cells were transfected with p3×Flag-CMV or Flag-PKM2 and ( A ) siPKM2 or siNC ( B ). Cell samples were analyzed at 24 and 48 h by immunoblotting with antibodies against AMPK, p-AMPK, p-mTOR, Flag, PKM2, and tubulin (loading control). ( C and D ) PK-15 and 3D4/2 cells were treated with 10 µM Compound C ( C ) or 10 µM AICAR ( D ). DMSO treatment was used as a control. Cell samples were analyzed at 24 h by immunoblotting with antibodies against AMPK and tubulin (loading control). Bar graphs represent the mean number of autophagosomes (puncta with both red and green colors, i.e., puncta with yellow color in merged images) and autolysosomes (puncta with only red but not green color, i.e., puncta with red color in merged images) per cell. Error bars indicate the mean (±SD) of three independent experiments. ns, P > 0.05; *** P < 0.001, and **** P < 0.0001 (two-way ANOVA). ( E ) PK-15 and 3D4/2 cells were treated with DMSO or Compound C and then transfected with p3×Flag-CMV or Flag-PKM2. Cell samples were analyzed at 24 h by immunoblotting with antibodies against Flag, p-mTOR, and tubulin (loading control). ( F ) PK-15 and 3D4/2 cells were treated with 10 µM DMSO or 10 µM AICAR and then transfected with siPKM2 or siNC. Cell samples were analyzed at 24 h by immunoblotting with antibodies against PKM2, p-mTOR, and tubulin (loading control). ( G ) PK-15 and 3D4/2 cells were transfected with the siPKM2 or siNC for 24 h and then mock infected or infected with CSFV (MOI = 1.0). Western blot was used to analyze the relative expression of proteins AMPK, p-AMPK, p-mTOR, and TUBA (loading control).

    Techniques Used: Infection, Transfection, Western Blot, Expressing

    PKM2 activated AMPK and enriched it in mitochondria. ( A ) The effect of FNZ (10 µM) on cell viability of PK-15 and 3D4/2 cells. Cells were treated with different concentrations of FNZ and evaluated by the CCK-8 assay. Error bars represent the mean ± SD; n = 3; ** P < 0.01; *** P < 0.001; **** P < 0.0001; and ns, P > 0.05. ( B ) PK-15 and 3D4/2 cells were treated with FNZ at different times. Western blot was used to analyze the relative expression of VDACI, COXIV, TOM20, HSP60, and tubulin (loading control). ( C ) PK-15 and 3D4/2 cells were transfected with p3×Flag-CMV or Flag-PKM2 and then treated with 10 µM FNZ, the same volume of DMSO. Western blot was used to analyze the relative expression of proteins AMPK, p-AMPK, p-mTOR, TOM20, COXIV, and tubulin (loading control). ( D ) Effect of PKM2 on AMPK mitochondrial translocation. PK-15 cells were transfected with P3×Flag-CMV or Flag-PKM2, followed by mitochondrial isolation using the Mitochondrial Isolation Kit. Western blot was used to analyze the relative expression of proteins AMPK, p-AMPK, p-mTOR, TOM20, COXIV, and tubulin (loading control). The level of protein was quantified using Image-Pro Plus 6.0 software. Error bars indicate the mean (±SD) of three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001; and **** P < 0.0001 (one-way ANOVA).
    Figure Legend Snippet: PKM2 activated AMPK and enriched it in mitochondria. ( A ) The effect of FNZ (10 µM) on cell viability of PK-15 and 3D4/2 cells. Cells were treated with different concentrations of FNZ and evaluated by the CCK-8 assay. Error bars represent the mean ± SD; n = 3; ** P < 0.01; *** P < 0.001; **** P < 0.0001; and ns, P > 0.05. ( B ) PK-15 and 3D4/2 cells were treated with FNZ at different times. Western blot was used to analyze the relative expression of VDACI, COXIV, TOM20, HSP60, and tubulin (loading control). ( C ) PK-15 and 3D4/2 cells were transfected with p3×Flag-CMV or Flag-PKM2 and then treated with 10 µM FNZ, the same volume of DMSO. Western blot was used to analyze the relative expression of proteins AMPK, p-AMPK, p-mTOR, TOM20, COXIV, and tubulin (loading control). ( D ) Effect of PKM2 on AMPK mitochondrial translocation. PK-15 cells were transfected with P3×Flag-CMV or Flag-PKM2, followed by mitochondrial isolation using the Mitochondrial Isolation Kit. Western blot was used to analyze the relative expression of proteins AMPK, p-AMPK, p-mTOR, TOM20, COXIV, and tubulin (loading control). The level of protein was quantified using Image-Pro Plus 6.0 software. Error bars indicate the mean (±SD) of three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001; and **** P < 0.0001 (one-way ANOVA).

    Techniques Used: CCK-8 Assay, Western Blot, Expressing, Transfection, Translocation Assay, Isolation, Software

    PKM2 regulated mitophagy through the AMPK-mTOR pathway. ( A ) PK-15 or 3D4/2 cells were pretreated with DMSO or Compound C (10 µM) for 2 h and then transfected with p3×Flag-CMV or Flag-PKM2. Cell samples were analyzed at 24 h by immunoblotting with antibodies against Flag, P62, COXIV, TOM20, LC3, and tubulin (loading control). ( B ) PK-15 or 3D4/2 cells were pretreated with DMSO or AICAR for 2 h and then transfected with siNC or siPKM2. Cell samples were analyzed at 24 h by immunoblotting with antibodies against PKM2, P62, COXIV, TOM20, LC3, and tubulin (loading control). The level of protein was quantified using Image-Pro Plus 6.0 software. Error bars indicate the mean (±SD) of three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001; and **** P < 0.0001 (one-way ANOVA). ( C ) PK-15 and 3D4/2 cells transiently expressing Mito-mRFP-EGFP were pretreated with DMSO/Compound C/Mdivi-1 (10 µM) for 2 h and then transfected with p3×Flag-CMV or Flag-PKM2. In the zoomed images, fluorescence signals indicated the expression of mRFP and GFP protein targeting mitochondria: yellow color, no mitophagy; red color, mitophagy. ( D ) PK-15 and 3D4/2 cells transiently expressing Mito-mRFP-EGFP were pretreated with DMSO/AICAR/CCCP (10 µM) for 2 h and then transfected with siNC or siPKM2. In the zoomed images, fluorescence signals indicated the expression of mRFP and GFP proteins targeting mitochondria: yellow color, no mitophagy; red color, mitophagy. Image-Pro Plus 6.0 software was used to measure the fluorescence intensity quantitatively. ns, P > 0.05; *** P < 0.001; and **** P < 0.0001 (two-way ANOVA).
    Figure Legend Snippet: PKM2 regulated mitophagy through the AMPK-mTOR pathway. ( A ) PK-15 or 3D4/2 cells were pretreated with DMSO or Compound C (10 µM) for 2 h and then transfected with p3×Flag-CMV or Flag-PKM2. Cell samples were analyzed at 24 h by immunoblotting with antibodies against Flag, P62, COXIV, TOM20, LC3, and tubulin (loading control). ( B ) PK-15 or 3D4/2 cells were pretreated with DMSO or AICAR for 2 h and then transfected with siNC or siPKM2. Cell samples were analyzed at 24 h by immunoblotting with antibodies against PKM2, P62, COXIV, TOM20, LC3, and tubulin (loading control). The level of protein was quantified using Image-Pro Plus 6.0 software. Error bars indicate the mean (±SD) of three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001; and **** P < 0.0001 (one-way ANOVA). ( C ) PK-15 and 3D4/2 cells transiently expressing Mito-mRFP-EGFP were pretreated with DMSO/Compound C/Mdivi-1 (10 µM) for 2 h and then transfected with p3×Flag-CMV or Flag-PKM2. In the zoomed images, fluorescence signals indicated the expression of mRFP and GFP protein targeting mitochondria: yellow color, no mitophagy; red color, mitophagy. ( D ) PK-15 and 3D4/2 cells transiently expressing Mito-mRFP-EGFP were pretreated with DMSO/AICAR/CCCP (10 µM) for 2 h and then transfected with siNC or siPKM2. In the zoomed images, fluorescence signals indicated the expression of mRFP and GFP proteins targeting mitochondria: yellow color, no mitophagy; red color, mitophagy. Image-Pro Plus 6.0 software was used to measure the fluorescence intensity quantitatively. ns, P > 0.05; *** P < 0.001; and **** P < 0.0001 (two-way ANOVA).

    Techniques Used: Transfection, Western Blot, Software, Expressing, Fluorescence

    PKM2 promoted CSFV proliferation via AMPK. (A and B) PK-15 or 3D4/2 cells were infected with CSFV (MOI = 1.0) for 2 h and treated with 10 µM AICAR ( A ) or Compound C ( B ), the same volume of DMSO. The relative expression of NS5B mRNA was detected by qRT-PCR at 24 h. ( C and D ) Cell treatment is the same as panel A. Cell samples were analyzed at 24 h by immunoblotting with antibodies against E2 and tubulin (loading control). ( E ) PK-15 or 3D4/2 cells were pretreated with DMSO or Compound C (10 µM) for 2 h and then transfected with p3×Flag-CMV or Flag-PKM2. Cell samples were analyzed at 24 h by immunoblotting with antibodies against Flag, E2, and tubulin (loading control). ( F ) PK-15 or 3D4/2 cells were pretreated with DMSO or AICAR (10 µM) for 2 h and then transfected with siNC or siPKM2. Cell samples were analyzed at 24 h by immunoblotting with antibodies against PKM2, E2, and tubulin (loading control). The level of protein was quantified using Image-Pro Plus 6.0 software. Error bars indicate the mean (±SD) of three independent experiments. ns, P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001; and **** P < 0.0001 (one-way ANOVA). ( G ) Cell treatment is the same as panel E . CSFV virus titers in the supernatant were determined as 50% tissue culture infective doses (TCID50)/mL as described in Materials and Methods. ( H ) Cell treatment is the same as panel F . CSFV virus titers in the supernatant were determined as TCID50/mL as described in Materials and Methods. Error bars indicate the mean (±SD) of three independent experiments. ns, P > 0.05; * P < 0.05; ** P < 0.01; and *** P < 0.001 (one-way ANOVA).
    Figure Legend Snippet: PKM2 promoted CSFV proliferation via AMPK. (A and B) PK-15 or 3D4/2 cells were infected with CSFV (MOI = 1.0) for 2 h and treated with 10 µM AICAR ( A ) or Compound C ( B ), the same volume of DMSO. The relative expression of NS5B mRNA was detected by qRT-PCR at 24 h. ( C and D ) Cell treatment is the same as panel A. Cell samples were analyzed at 24 h by immunoblotting with antibodies against E2 and tubulin (loading control). ( E ) PK-15 or 3D4/2 cells were pretreated with DMSO or Compound C (10 µM) for 2 h and then transfected with p3×Flag-CMV or Flag-PKM2. Cell samples were analyzed at 24 h by immunoblotting with antibodies against Flag, E2, and tubulin (loading control). ( F ) PK-15 or 3D4/2 cells were pretreated with DMSO or AICAR (10 µM) for 2 h and then transfected with siNC or siPKM2. Cell samples were analyzed at 24 h by immunoblotting with antibodies against PKM2, E2, and tubulin (loading control). The level of protein was quantified using Image-Pro Plus 6.0 software. Error bars indicate the mean (±SD) of three independent experiments. ns, P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001; and **** P < 0.0001 (one-way ANOVA). ( G ) Cell treatment is the same as panel E . CSFV virus titers in the supernatant were determined as 50% tissue culture infective doses (TCID50)/mL as described in Materials and Methods. ( H ) Cell treatment is the same as panel F . CSFV virus titers in the supernatant were determined as TCID50/mL as described in Materials and Methods. Error bars indicate the mean (±SD) of three independent experiments. ns, P > 0.05; * P < 0.05; ** P < 0.01; and *** P < 0.001 (one-way ANOVA).

    Techniques Used: Infection, Expressing, Quantitative RT-PCR, Western Blot, Transfection, Software, Virus

    Overexpression of PKM2 promoted CSFV proliferation via mitophagy. ( A and B ) PK-15 or 3D4/2 cells were infected with CSFV (MOI = 1.0) for 2 h and treated with 10 µM Mdivi-1, the same volume of DMSO. Cell samples were analyzed at 24 h by immunoblotting with antibodies against E2 and tubulin (loading control). The level of protein was quantified using Image-Pro Plus 6.0 software. Error bars indicate the mean (±SD) of three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001; and **** P < 0.0001 (one-way ANOVA). ( C and D ) Cell treatment is the same as panel A. CSFV virus titers in the supernatant were determined as 50% tissue culture infective doses (TCID50)/mL as described in Materials and Methods. Error bars represent the mean ± SD; n = 3; * P < 0.05; ** P < 0.01; *** P < 0.001; and ns, P > 0.05 (one-way ANOVA).
    Figure Legend Snippet: Overexpression of PKM2 promoted CSFV proliferation via mitophagy. ( A and B ) PK-15 or 3D4/2 cells were infected with CSFV (MOI = 1.0) for 2 h and treated with 10 µM Mdivi-1, the same volume of DMSO. Cell samples were analyzed at 24 h by immunoblotting with antibodies against E2 and tubulin (loading control). The level of protein was quantified using Image-Pro Plus 6.0 software. Error bars indicate the mean (±SD) of three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001; and **** P < 0.0001 (one-way ANOVA). ( C and D ) Cell treatment is the same as panel A. CSFV virus titers in the supernatant were determined as 50% tissue culture infective doses (TCID50)/mL as described in Materials and Methods. Error bars represent the mean ± SD; n = 3; * P < 0.05; ** P < 0.01; *** P < 0.001; and ns, P > 0.05 (one-way ANOVA).

    Techniques Used: Over Expression, Infection, Western Blot, Software, Virus

    The effect of RNA interference on cell viability. The cell viability of PK-15 (A) cells and 3D4/2 (B) cells transfected with siNC or PKM2 siRNA-1/-2/-3 was analyzed using the CCK8 assay as described in Materials and Methods (mean ± SD; n = 3; ns, P > 0.05).
    Figure Legend Snippet: The effect of RNA interference on cell viability. The cell viability of PK-15 (A) cells and 3D4/2 (B) cells transfected with siNC or PKM2 siRNA-1/-2/-3 was analyzed using the CCK8 assay as described in Materials and Methods (mean ± SD; n = 3; ns, P > 0.05).

    Techniques Used: Transfection, CCK-8 Assay

    swine kidney cell line pk 15  (ATCC)


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    swine kidney cell line pk 15  (ATCC)


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    ATCC swine kidney cell line pk 15
    Necroptosis was induced at the early stage and inhibited at the late stage of CSFV infection in vitro . To establish a PBMC cell model of CSFV infection in vitro , cells were collected at 24, 48, and 60 h post-infection, and protein expression and mRNA levels of necroptosis markers including RIPK1, RIPK3, MLKL, and ZBP1 were detected by Western blot ( A ) and qRT-PCR ( G ). ( B ) Gray-scale value analysis of the detection indexes in ( A ); the relative intensity was related to the internal control Tubulin. Error bars indicate the mean (± SD) of three independent experiments. ns, P ≥ 0.05*; P < 0.05; **, P < 0.01; and ***, P < 0.001 (two-way ANOVA). To establish a <t>PK-15</t> cell model of CSFV infection in vitro , cells were collected at 24, 48, and 60 h post-infection, and protein expression and mRNA levels of necroptosis marker genes including RIPK1, RIPK3, MLKL, and ZBP1 were detected by Western blot ( C ) and qRT-PCR ( H ). ( D ) Gray-scale value analysis of the detection indexes in ( C ); the relative intensity was related to the internal control Tubulin. Error bars indicate the mean (± SD) of three independent experiments. ns, P ≥ 0.05*; P < 0.05; **, P < 0.01; and ***, P < 0.001 (two-way ANOVA). To establish a 3D4/21 cell model of CSFV infection in vitro , cells were collected at 24, 48, and 60 h post-infection, and protein expression and mRNA levels of necroptosis markers including RIPK1, RIPK3, MLKL, and ZBP1 were detected by Western blot ( E ) and qRT-PCR ( I ). ( F ) Gray-scale value analysis of the detection indexes in ( E ); the relative intensity was related to the internal control Tubulin. Error bars indicate the mean (± SD) of three independent experiments. ns, P ≥ 0.05*; P < 0.05; **, P < 0.01; and ***, P < 0.001 (two-way ANOVA).
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    1) Product Images from "CSFV restricts necroptosis to sustain infection by inducing autophagy/mitophagy-targeted degradation of RIPK3"

    Article Title: CSFV restricts necroptosis to sustain infection by inducing autophagy/mitophagy-targeted degradation of RIPK3

    Journal: Microbiology Spectrum

    doi: 10.1128/spectrum.02758-23

    Necroptosis was induced at the early stage and inhibited at the late stage of CSFV infection in vitro . To establish a PBMC cell model of CSFV infection in vitro , cells were collected at 24, 48, and 60 h post-infection, and protein expression and mRNA levels of necroptosis markers including RIPK1, RIPK3, MLKL, and ZBP1 were detected by Western blot ( A ) and qRT-PCR ( G ). ( B ) Gray-scale value analysis of the detection indexes in ( A ); the relative intensity was related to the internal control Tubulin. Error bars indicate the mean (± SD) of three independent experiments. ns, P ≥ 0.05*; P < 0.05; **, P < 0.01; and ***, P < 0.001 (two-way ANOVA). To establish a PK-15 cell model of CSFV infection in vitro , cells were collected at 24, 48, and 60 h post-infection, and protein expression and mRNA levels of necroptosis marker genes including RIPK1, RIPK3, MLKL, and ZBP1 were detected by Western blot ( C ) and qRT-PCR ( H ). ( D ) Gray-scale value analysis of the detection indexes in ( C ); the relative intensity was related to the internal control Tubulin. Error bars indicate the mean (± SD) of three independent experiments. ns, P ≥ 0.05*; P < 0.05; **, P < 0.01; and ***, P < 0.001 (two-way ANOVA). To establish a 3D4/21 cell model of CSFV infection in vitro , cells were collected at 24, 48, and 60 h post-infection, and protein expression and mRNA levels of necroptosis markers including RIPK1, RIPK3, MLKL, and ZBP1 were detected by Western blot ( E ) and qRT-PCR ( I ). ( F ) Gray-scale value analysis of the detection indexes in ( E ); the relative intensity was related to the internal control Tubulin. Error bars indicate the mean (± SD) of three independent experiments. ns, P ≥ 0.05*; P < 0.05; **, P < 0.01; and ***, P < 0.001 (two-way ANOVA).
    Figure Legend Snippet: Necroptosis was induced at the early stage and inhibited at the late stage of CSFV infection in vitro . To establish a PBMC cell model of CSFV infection in vitro , cells were collected at 24, 48, and 60 h post-infection, and protein expression and mRNA levels of necroptosis markers including RIPK1, RIPK3, MLKL, and ZBP1 were detected by Western blot ( A ) and qRT-PCR ( G ). ( B ) Gray-scale value analysis of the detection indexes in ( A ); the relative intensity was related to the internal control Tubulin. Error bars indicate the mean (± SD) of three independent experiments. ns, P ≥ 0.05*; P < 0.05; **, P < 0.01; and ***, P < 0.001 (two-way ANOVA). To establish a PK-15 cell model of CSFV infection in vitro , cells were collected at 24, 48, and 60 h post-infection, and protein expression and mRNA levels of necroptosis marker genes including RIPK1, RIPK3, MLKL, and ZBP1 were detected by Western blot ( C ) and qRT-PCR ( H ). ( D ) Gray-scale value analysis of the detection indexes in ( C ); the relative intensity was related to the internal control Tubulin. Error bars indicate the mean (± SD) of three independent experiments. ns, P ≥ 0.05*; P < 0.05; **, P < 0.01; and ***, P < 0.001 (two-way ANOVA). To establish a 3D4/21 cell model of CSFV infection in vitro , cells were collected at 24, 48, and 60 h post-infection, and protein expression and mRNA levels of necroptosis markers including RIPK1, RIPK3, MLKL, and ZBP1 were detected by Western blot ( E ) and qRT-PCR ( I ). ( F ) Gray-scale value analysis of the detection indexes in ( E ); the relative intensity was related to the internal control Tubulin. Error bars indicate the mean (± SD) of three independent experiments. ns, P ≥ 0.05*; P < 0.05; **, P < 0.01; and ***, P < 0.001 (two-way ANOVA).

    Techniques Used: Infection, In Vitro, Expressing, Western Blot, Quantitative RT-PCR, Marker

    CSFV NS4A interacts with RIPK3 and TRIM25 to inhibit necroptosis. ( A ) HEK-293T cells were co-transfected with HA-RIPK3 together with pGFP-C1, GFP-P7, GFP-C, GFP-NS4A, GFP-NS4B, or GFP-NS5A for 24 h, fixed cells were incubated with anti-HA tag primary antibody and then stained with Alexa fluor 555-conjugated anti-Rabbit-IgG secondary antibody (red), and nuclei were stained with DAPI. Scale bars: 25 µm. ( B ) pGFP-C1, GFP-P7, GFP-C, GFP-NS4A, GFP-NS4B, and GFP-NS5A were transfected into PK-15 cells for 24 h, and the cells were lysed. The expression levels of viral proteins, RIPK1, RIPK3, and MLKL, were detected by Western blot. The right panel was gray-scale value analysis, the relative intensity was related to the internal control GAPDH. Error bars indicate the mean (± SD) of three independent experiments. ****, P < 0.0001 (two-way ANOVA). ( C ) FLAG-RIPK3 was co-transfected with pEGFP-C1 and GFP-NS4A, respectively, into PK-15 cells, and the cells were lysed; Co-IP assay and Western blot analysis were performed. ( D ) Myc-TRIM25 was co-transfected with pEGFP-C1 and GFP-NS4A in HEK-293T cells, while a control group of cells was set up in which pCMV-N1-Myc was co-transfected with pEGFP-C1 and GFP-NS4A, cells were lysed; Co-IP experiments and Western blot analysis were performed. ( E ) PK-15 cells were co-transfected with three constructs, HA-RIPK3, Myc-TRIM25 with GFP-NS4A, and a control construct of cells co-transfected with HA-RIPK3, Myc-TRIM25 with pEGFP-C1, HA-RIPK3, pCMV-N1-Myc with GFP-NS4A, Myc-TRIM25, pCAGGs-HA with GFP-NS4A, The fixed cells were incubated with anti-HA primary antibody and anti-Myc primary antibody, respectively, and then stained with Alexa fluor 555-conjugated anti-Rabbit-IgG Mouse antibody (red) and Dylight 405-labeled anti-Rabbit-IgG secondary antibody (blue) (left panel). Scale bars: 10 µm. The fluorescence intensity profiles of HA-RIPK3 (blue), GFP-NS4A(green), and Myc-TRIM25(red) were analyzed using Image J plugin and plotted by GraphPad Prism 9 (right panel). ( F ) Myc-TRIM25 was co-transfected with pEGFP-C1 and GFP-NS4A in PK-15 cells, while a control group of cells was set up in which pCMV-N1-Myc was co-transfected with pEGFP-C1 and GFP-NS4A; the cell lysates were detected by Western blot. The gray-scale value analysis was related to the internal control GAPDH (lower panel). Error bars indicate the mean (± SD) of three independent experiments. ****, P < 0.0001 (two-way ANOVA).
    Figure Legend Snippet: CSFV NS4A interacts with RIPK3 and TRIM25 to inhibit necroptosis. ( A ) HEK-293T cells were co-transfected with HA-RIPK3 together with pGFP-C1, GFP-P7, GFP-C, GFP-NS4A, GFP-NS4B, or GFP-NS5A for 24 h, fixed cells were incubated with anti-HA tag primary antibody and then stained with Alexa fluor 555-conjugated anti-Rabbit-IgG secondary antibody (red), and nuclei were stained with DAPI. Scale bars: 25 µm. ( B ) pGFP-C1, GFP-P7, GFP-C, GFP-NS4A, GFP-NS4B, and GFP-NS5A were transfected into PK-15 cells for 24 h, and the cells were lysed. The expression levels of viral proteins, RIPK1, RIPK3, and MLKL, were detected by Western blot. The right panel was gray-scale value analysis, the relative intensity was related to the internal control GAPDH. Error bars indicate the mean (± SD) of three independent experiments. ****, P < 0.0001 (two-way ANOVA). ( C ) FLAG-RIPK3 was co-transfected with pEGFP-C1 and GFP-NS4A, respectively, into PK-15 cells, and the cells were lysed; Co-IP assay and Western blot analysis were performed. ( D ) Myc-TRIM25 was co-transfected with pEGFP-C1 and GFP-NS4A in HEK-293T cells, while a control group of cells was set up in which pCMV-N1-Myc was co-transfected with pEGFP-C1 and GFP-NS4A, cells were lysed; Co-IP experiments and Western blot analysis were performed. ( E ) PK-15 cells were co-transfected with three constructs, HA-RIPK3, Myc-TRIM25 with GFP-NS4A, and a control construct of cells co-transfected with HA-RIPK3, Myc-TRIM25 with pEGFP-C1, HA-RIPK3, pCMV-N1-Myc with GFP-NS4A, Myc-TRIM25, pCAGGs-HA with GFP-NS4A, The fixed cells were incubated with anti-HA primary antibody and anti-Myc primary antibody, respectively, and then stained with Alexa fluor 555-conjugated anti-Rabbit-IgG Mouse antibody (red) and Dylight 405-labeled anti-Rabbit-IgG secondary antibody (blue) (left panel). Scale bars: 10 µm. The fluorescence intensity profiles of HA-RIPK3 (blue), GFP-NS4A(green), and Myc-TRIM25(red) were analyzed using Image J plugin and plotted by GraphPad Prism 9 (right panel). ( F ) Myc-TRIM25 was co-transfected with pEGFP-C1 and GFP-NS4A in PK-15 cells, while a control group of cells was set up in which pCMV-N1-Myc was co-transfected with pEGFP-C1 and GFP-NS4A; the cell lysates were detected by Western blot. The gray-scale value analysis was related to the internal control GAPDH (lower panel). Error bars indicate the mean (± SD) of three independent experiments. ****, P < 0.0001 (two-way ANOVA).

    Techniques Used: Transfection, Incubation, Staining, Expressing, Western Blot, Co-Immunoprecipitation Assay, Construct, Labeling, Fluorescence

    CSFV inhibits necroptosis through autophagy/mitophagy and necroptosis-restricted CSFV infection in vitro . ( A ) PK-15 cells were infected with CSFV (MOI = 1) for 48 h, rapamycin was treated for 6 h, and an uninfected cell control was set up. Cell lysates were then detected by Western blot (left panel). Relative intensity related to internal control Tubulin was analyzed using Image J plugin and plotted by GraphPad Prism 9 (right panel). Error bars indicate the mean (± SD) of three independent experiments. ns, P ≥ 0.05; *, P < 0. 1; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (two-way ANOVA). ( B ) PK-15 cells were infected with CSFV (MOI = 1) for 48 h, CCCP treated for 6 h, and an uninfected cell control was set up. Cell lysates were then detected by Western blot (left panel). Relative intensity related to internal control Tubulin was analyzed using Image J plugin and plotted by GraphPad Prism 9 (right panel). Error bars indicate the mean (± SD) of three independent experiments. ns, P ≥ 0.05; *, P < 0. 1; ***, P < 0.001; ****, P < 0.0001 (two-way ANOVA). ( C ) PK-15 cells were infected with CSFV (MOI = 1) for 48 h and treated with TSZ (20 ng/mL TNF-α, 100 nM Smac mimetic, and 20 µM z-VAD-Fmk) alone or TSZ together with rapamycin for 6 h, and a control of uninfected cells was set up. Necrotic cells were identified by flow cytometry with PI and Annexin V staining. A representative plot of the data is shown in the left panel, and the mean (± SD) of three independent experiments is shown in the right panel. *, P < 0. 1 and ****, P < 0.0001 (two-way ANOVA). ( D ) PK-15 cells infected with CSFV (MOI = 1) were transfected with Flag-RIPK3 for 24 h and then treated with 3-methyladenine (3-MA, 5 mM), chloroquine (CQ, 20 µM), or bafilomycin A1 (Baf A1, 100 nM) for 6 h, and cell lysates were detected by Western blot (upper panel). Flag-RIPK3 was co-transfected with shNC, shATG5, shBECN1, and shLC3 into CSFV-infected PK-15 cells, and cell lysates were detected by Western blot (lower panel). ( E ) PK-15 (left panel) and 3D4/21 (right panel) cells infected with CSFV (MOI = 1) were transfected with HA-RIPK3 or MLKL for 48 h, and pCAGGs-HA was transfected with a control group. The cell supernatants were collected for TCID50 assay. Error bars indicate the mean (± SD) of three independent experiments. *, P < 0.1 and **, P < 0.01 (one-way ANOVA). ( F ) PK-15 cells infected with CSFV (MOI = 1) were treated with TSZ for 6 h, and control of uninfected cells was set up. The mRNA expressions of RIPK1 , RIPK3 , MLKL, and CSFV NS5B in the cell lysates were determined by qRT-PCR. The fold changes were related to the internal control β-actin. Error bars indicate the mean (± SD) of three independent experiments. *, P < 0.1; **, P < 0.01; ***, P < 0.001 (one-way ANOVA). ( G ) PK-15 cells infected with CSFV (MOI = 1) were treated with increasing amounts of TSZ (wedge shaped, 0; 20 ng/mL TNF-α, 100 nM Smac mimetic, and 10 µM z-VAD-Fmk; 40 ng/mL TNF-α, 100 nM Smac mimetic, and 10 µM z-VAD-Fmk; 40 ng/mL TNF-α, 100 nM Smac mimetic, and 20 µM z-VAD-Fmk) for 6 h, and the cell lysates were detected by Western blot. ( H ) PK-15 cells were treated with increasing amounts of necrosulfonamide (NSA; wedge shaped, 0, 1, 5, and 10 µM), and the cell lysates were detected by Western blot (upper panel). PK-15 cells infected with CSFV (MOI = 1) were treated with NSA (5 µM), and dimethyl sulfoxide (DMSO) was treated with a control group. The cell lysates were detected by Western blot (lower panel). ( I ) PK-15 (left panel) and 3D4/21 (right panel) cells were infected with CSFV (MOI = 1) for 48 h and treated with TSZ (20 ng/mL TNF-α, 100 nM Smac mimetic, and 20 µM z-VAD -Fmk) alone or TSZ together with rapamycin for 6 h, and a control of uninfected cells was set up. The cell lysates were detected by Western blot.
    Figure Legend Snippet: CSFV inhibits necroptosis through autophagy/mitophagy and necroptosis-restricted CSFV infection in vitro . ( A ) PK-15 cells were infected with CSFV (MOI = 1) for 48 h, rapamycin was treated for 6 h, and an uninfected cell control was set up. Cell lysates were then detected by Western blot (left panel). Relative intensity related to internal control Tubulin was analyzed using Image J plugin and plotted by GraphPad Prism 9 (right panel). Error bars indicate the mean (± SD) of three independent experiments. ns, P ≥ 0.05; *, P < 0. 1; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (two-way ANOVA). ( B ) PK-15 cells were infected with CSFV (MOI = 1) for 48 h, CCCP treated for 6 h, and an uninfected cell control was set up. Cell lysates were then detected by Western blot (left panel). Relative intensity related to internal control Tubulin was analyzed using Image J plugin and plotted by GraphPad Prism 9 (right panel). Error bars indicate the mean (± SD) of three independent experiments. ns, P ≥ 0.05; *, P < 0. 1; ***, P < 0.001; ****, P < 0.0001 (two-way ANOVA). ( C ) PK-15 cells were infected with CSFV (MOI = 1) for 48 h and treated with TSZ (20 ng/mL TNF-α, 100 nM Smac mimetic, and 20 µM z-VAD-Fmk) alone or TSZ together with rapamycin for 6 h, and a control of uninfected cells was set up. Necrotic cells were identified by flow cytometry with PI and Annexin V staining. A representative plot of the data is shown in the left panel, and the mean (± SD) of three independent experiments is shown in the right panel. *, P < 0. 1 and ****, P < 0.0001 (two-way ANOVA). ( D ) PK-15 cells infected with CSFV (MOI = 1) were transfected with Flag-RIPK3 for 24 h and then treated with 3-methyladenine (3-MA, 5 mM), chloroquine (CQ, 20 µM), or bafilomycin A1 (Baf A1, 100 nM) for 6 h, and cell lysates were detected by Western blot (upper panel). Flag-RIPK3 was co-transfected with shNC, shATG5, shBECN1, and shLC3 into CSFV-infected PK-15 cells, and cell lysates were detected by Western blot (lower panel). ( E ) PK-15 (left panel) and 3D4/21 (right panel) cells infected with CSFV (MOI = 1) were transfected with HA-RIPK3 or MLKL for 48 h, and pCAGGs-HA was transfected with a control group. The cell supernatants were collected for TCID50 assay. Error bars indicate the mean (± SD) of three independent experiments. *, P < 0.1 and **, P < 0.01 (one-way ANOVA). ( F ) PK-15 cells infected with CSFV (MOI = 1) were treated with TSZ for 6 h, and control of uninfected cells was set up. The mRNA expressions of RIPK1 , RIPK3 , MLKL, and CSFV NS5B in the cell lysates were determined by qRT-PCR. The fold changes were related to the internal control β-actin. Error bars indicate the mean (± SD) of three independent experiments. *, P < 0.1; **, P < 0.01; ***, P < 0.001 (one-way ANOVA). ( G ) PK-15 cells infected with CSFV (MOI = 1) were treated with increasing amounts of TSZ (wedge shaped, 0; 20 ng/mL TNF-α, 100 nM Smac mimetic, and 10 µM z-VAD-Fmk; 40 ng/mL TNF-α, 100 nM Smac mimetic, and 10 µM z-VAD-Fmk; 40 ng/mL TNF-α, 100 nM Smac mimetic, and 20 µM z-VAD-Fmk) for 6 h, and the cell lysates were detected by Western blot. ( H ) PK-15 cells were treated with increasing amounts of necrosulfonamide (NSA; wedge shaped, 0, 1, 5, and 10 µM), and the cell lysates were detected by Western blot (upper panel). PK-15 cells infected with CSFV (MOI = 1) were treated with NSA (5 µM), and dimethyl sulfoxide (DMSO) was treated with a control group. The cell lysates were detected by Western blot (lower panel). ( I ) PK-15 (left panel) and 3D4/21 (right panel) cells were infected with CSFV (MOI = 1) for 48 h and treated with TSZ (20 ng/mL TNF-α, 100 nM Smac mimetic, and 20 µM z-VAD -Fmk) alone or TSZ together with rapamycin for 6 h, and a control of uninfected cells was set up. The cell lysates were detected by Western blot.

    Techniques Used: Infection, In Vitro, Western Blot, Flow Cytometry, Staining, Transfection, TCID50 Assay, Quantitative RT-PCR

    CSFV NS4A synergizes TRIM25 to induce mitophagy. ( A and B ) PK-15 and 3D4/21 cells were transfected with Myc-TRIM25 for 24 h, and pCMV-N1-Myc cells were transfected with control group. Expression levels of autophagy-associated proteins p-mTOR, LC3-II, and P62 and mitochondrial-associated proteins HSP60, TOMM20, COX IV, and VDAC in cell lysis products were detected by Western blot (left panel). Relative intensity related to internal parameters was analyzed using Image J plugin and plotted by GraphPad Prism 9 (right panel). Error bars indicate the mean (± SD) of three independent experiments. ns, P ≥ 0.05; **, P < 0.01; ***, P < 0.001 (two-way ANOVA). ( C ) PK-15 and 3D4/21 cells were transfected with Myc-TRIM25 for 24 h, and pCMV-N1-Myc cells were set up as a control group. The cells were immunostained with Mito-Tracker, incubated with anti-Myc primary antibody, followed by Alexa fluor 488-conjugated anti-Rabbit-IgG secondary antibody (green), and the nuclei were stained with DAPI. Scale bars: 25 and 10 µm. ( D ) Mitochondrial structures and autophagy-like vesicles were visualized by transmission electron microscopy (left panel), and the number of autophagic vesicles with encapsulated mitochondria was quantified (right panel). Error bars indicate the mean (± SD) of five independent experiments. ***, P < 0.0005 (one-way ANOVA). ( E ) Myc-TRIM25 was co-transfected with pEGFP-C1 and GFP-NS4A in PK-15 cells, respectively, while a control group of cells was set up in which pCMV-N1-Myc was co-transfected with pEGFP-C1 and GFP-NS4A, respectively. Expression levels of autophagy-associated proteins p-mTOR, LC3-II, and P62 and mitochondrial-associated proteins HSP60, TOMM20, COX IV, and VDAC in cell lysis products were detected by Western blot. ( F ) The relative intensity of the detection indexes in ( E ), related to the internal control GAPDH, was analyzed with Image J plugin and plotted by GraphPad Prism 9. Error bars indicate the mean (± SD) of three independent experiments. *, P < 0.1; and ****, P < 0.0001 (two-way ANOVA). ( G ) Si TRIM25 was co-transfected with pEGFP-C1 and GFP-NS4A in PK-15 cells, respectively, while a control group of cells was set up in which Si NC was co-transfected with pEGFP-C1 and GFP-NS4A, respectively. Expression levels of autophagy-associated proteins p-mTOR, LC3-II, and P62 and mitochondrial-associated proteins HSP60, TOMM20, COX IV, and VDAC in cell lysis products were detected by Western blot. ( H ) The relative intensity of the detection indexes in ( G ), related to the internal control GAPDH, was analyzed with Image J plugin and plotted by GraphPad Prism 9. Error bars indicate the mean (± SD) of three independent experiments. ns, P ≥ 0.05 and ****, P < 0.0001 (two-way ANOVA). ( I ) HA-NS4A was co-transfected with Myc-TRIM25 and Si TRIM25 into PK-15 cells, respectively, and a cell control transfected with HA-NS4A only and pCAGGs-HA only was set up, along with dual-fluorescent reporter plasmid mRFP-GFP-LC3 to visualize the autophagic flux. ( J ) HA-NS4A was co-transfected with Myc-TRIM25 and Si TRIM25 into PK-15 cells, respectively, and a cell control transfected with HA-NS4A only and pCAGGs-HA only was set up, along with mitophagy dual-fluorescent reporter plasmid Mito-mRFP-EGFP to visualize the mitochondria-lysosome delivery. The fluorescence intensity of GFP-Mito (green) and RFP-Mito (red) was analyzed in Fig. S4E with Image J plugin and plotted by GraphPad Prism 9. Error bars indicate the mean (± SD) of at least three independent experiments. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001 (two-way ANOVA).
    Figure Legend Snippet: CSFV NS4A synergizes TRIM25 to induce mitophagy. ( A and B ) PK-15 and 3D4/21 cells were transfected with Myc-TRIM25 for 24 h, and pCMV-N1-Myc cells were transfected with control group. Expression levels of autophagy-associated proteins p-mTOR, LC3-II, and P62 and mitochondrial-associated proteins HSP60, TOMM20, COX IV, and VDAC in cell lysis products were detected by Western blot (left panel). Relative intensity related to internal parameters was analyzed using Image J plugin and plotted by GraphPad Prism 9 (right panel). Error bars indicate the mean (± SD) of three independent experiments. ns, P ≥ 0.05; **, P < 0.01; ***, P < 0.001 (two-way ANOVA). ( C ) PK-15 and 3D4/21 cells were transfected with Myc-TRIM25 for 24 h, and pCMV-N1-Myc cells were set up as a control group. The cells were immunostained with Mito-Tracker, incubated with anti-Myc primary antibody, followed by Alexa fluor 488-conjugated anti-Rabbit-IgG secondary antibody (green), and the nuclei were stained with DAPI. Scale bars: 25 and 10 µm. ( D ) Mitochondrial structures and autophagy-like vesicles were visualized by transmission electron microscopy (left panel), and the number of autophagic vesicles with encapsulated mitochondria was quantified (right panel). Error bars indicate the mean (± SD) of five independent experiments. ***, P < 0.0005 (one-way ANOVA). ( E ) Myc-TRIM25 was co-transfected with pEGFP-C1 and GFP-NS4A in PK-15 cells, respectively, while a control group of cells was set up in which pCMV-N1-Myc was co-transfected with pEGFP-C1 and GFP-NS4A, respectively. Expression levels of autophagy-associated proteins p-mTOR, LC3-II, and P62 and mitochondrial-associated proteins HSP60, TOMM20, COX IV, and VDAC in cell lysis products were detected by Western blot. ( F ) The relative intensity of the detection indexes in ( E ), related to the internal control GAPDH, was analyzed with Image J plugin and plotted by GraphPad Prism 9. Error bars indicate the mean (± SD) of three independent experiments. *, P < 0.1; and ****, P < 0.0001 (two-way ANOVA). ( G ) Si TRIM25 was co-transfected with pEGFP-C1 and GFP-NS4A in PK-15 cells, respectively, while a control group of cells was set up in which Si NC was co-transfected with pEGFP-C1 and GFP-NS4A, respectively. Expression levels of autophagy-associated proteins p-mTOR, LC3-II, and P62 and mitochondrial-associated proteins HSP60, TOMM20, COX IV, and VDAC in cell lysis products were detected by Western blot. ( H ) The relative intensity of the detection indexes in ( G ), related to the internal control GAPDH, was analyzed with Image J plugin and plotted by GraphPad Prism 9. Error bars indicate the mean (± SD) of three independent experiments. ns, P ≥ 0.05 and ****, P < 0.0001 (two-way ANOVA). ( I ) HA-NS4A was co-transfected with Myc-TRIM25 and Si TRIM25 into PK-15 cells, respectively, and a cell control transfected with HA-NS4A only and pCAGGs-HA only was set up, along with dual-fluorescent reporter plasmid mRFP-GFP-LC3 to visualize the autophagic flux. ( J ) HA-NS4A was co-transfected with Myc-TRIM25 and Si TRIM25 into PK-15 cells, respectively, and a cell control transfected with HA-NS4A only and pCAGGs-HA only was set up, along with mitophagy dual-fluorescent reporter plasmid Mito-mRFP-EGFP to visualize the mitochondria-lysosome delivery. The fluorescence intensity of GFP-Mito (green) and RFP-Mito (red) was analyzed in Fig. S4E with Image J plugin and plotted by GraphPad Prism 9. Error bars indicate the mean (± SD) of at least three independent experiments. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001 (two-way ANOVA).

    Techniques Used: Transfection, Expressing, Lysis, Western Blot, Incubation, Staining, Transmission Assay, Electron Microscopy, Plasmid Preparation, Fluorescence

    RIPK3 is able to be degraded by TRIM25-induced mitophagy under CSFV infection. ( A ) PK-15 cells infected with CSFV (MOI = 1) were co-transfected with Myc-TRIM25 and HA-RIPK3 for 24 h and then treated with MG132 (10 mM), rapamycin (100 nM), or Baf A1 (100 nM) for 6 h, and cell lysates were detected by Western blot (left panel). Relative intensity related to internal control GAPDH was analyzed using Image J plugin and plotted by GraphPad Prism 9 (right panel). Error bars indicate the mean (± SD) of three independent experiments. ns, P ≥ 0.05 and ****, P < 0.0001 (one-way ANOVA). ( B ) PK-15 cells infected with CSFV (MOI = 1) were co-transfected with Myc-TRIM25, Flag-RIPK3, and His-Ub for 24 h and then treated with MG132 (10 mM) for 6 h, and the cells were lysed and used for IP experiments. ( C ) HA-RIPK3 and GFP-LC3 were co-transfected into PK-15 cells for 24 h and then treated with rapamycin (100 nM) for 6 h while setting up a control group of cells treated with dimethyl sulfoxide (DMSO). The cells were incubated with anti-HA primary antibody, followed by Alexa fluor 555-conjugated anti-Rabbit-IgG secondary antibody (red), and the nuclei were stained with DAPI (left panel). Scale bars: 100 and 25 µm. The fluorescence intensity profiles of GFP-LC3 (green) and HA-RIPK3 (red) were analyzed using Image J plugin and plotted by GraphPad Prism 9 (right panel). ( D ) PK-15 cells were co-transfected with HA-RIPK3 together with Si TRIM25 for 24 h while setting up a control group of co-transfected Si NC cells, the cells were immunostained with Mito-Tracker, incubated with anti-HA primary antibody, followed by Alexa fluor 488-conjugated anti-Mouse-IgG secondary antibody (green), and nuclei were stained with DAPI (left panel). Scale bars: 100 and 10 µm. The fluorescence intensity profiles of HA-RIPK3 (green) and Mito-Tracker (red) were analyzed using Image J plugin and plotted by GraphPad Prism 9 (middle panel). Pearson’s correlation and overlap co-efficient of HA-RIPK3 and Mito-Tracker were analyzed using Image J plugin and plotted by GraphPad Prism 9 (right panel). Error bars indicate the mean (± SD) of at least three independent experiments. ns, P ≥ 0.05 and *, P < 0.1 (one-way ANOVA). ( E ) PK-15 cells infected with CSFV (MOI = 1) were co-transfected with Si TRIM25, Flag-RIPK3, and GFP-LC3 for 24 h while setting up a control group of co-transfected Si NC cells, the fixed cells were incubated with anti-Flag primary antibody and anti-CD63 primary antibody, respectively, and then stained with Alexa fluor 555-conjugated anti-Mouse-IgG secondary antibody (red) and Dylight 405-labeled anti-Rabbit-IgG secondary antibody (blue) (left panel). Scale bars: 10 µm. The fluorescence intensity profiles of Flag-RIPK3 (blue), GFP-LC3(green), and CD63(red) were analyzed using Image J plugin and plotted by GraphPad Prism 9 (middle panel). Pearson’s correlation and overlap co-efficient were analyzed using Image J plugin and plotted by GraphPad Prism 9 (right panel). Error bars indicate the mean (± SD) of at least three independent experiments. ***, P < 0.001 and ****, P < 0.0001 (one-way ANOVA). ( F ) The mRNA expressions of TRIM25 in the tonsil, lung, lymph node, spleen, kidney, and thymus of CSFV-infected and non-infected piglets were determined by qRT-PCR. The fold changes were related to the internal control β-actin. Error bars indicate the mean (± SD) of three independent experiments. ****, P < 0.0001 (two-way ANOVA). ( G ) To establish PK-15 (upper panel) and 3D4/21 (lower panel) cell models of CSFV infection in vitro , cells were collected at 24 and 48 h post-infection, and protein expression levels of TRIM25 and CSFV N pro were detected by Western blot. ( H ) PK-15 (left panel) and 3D4/21 (right panel) cells infected with CSFV (MOI = 1) were transfected with Myc-TRIM25 or Si TRIM25 for 48 h, and pCMV-N1-Myc or Si NC cells were transfected with a control group. The cell supernatants were collected for TCID 50 assay. Error bars indicate the mean (± SD) of three independent experiments. *, P < 0.1; ***, P < 0.001; ****, P < 0.0001 (one-way ANOVA). ( I ) PK-15 (left panel) and 3D4/21 (right panel) cells infected with CSFV (MOI = 1) were transfected with Myc-TRIM25 for 24 and 48 h, and pCMV-N1-Myc cells were transfected with a control group. Expression levels of TRIM25 and CSFV E2/N pro in cell lysis products were detected by Western blot.
    Figure Legend Snippet: RIPK3 is able to be degraded by TRIM25-induced mitophagy under CSFV infection. ( A ) PK-15 cells infected with CSFV (MOI = 1) were co-transfected with Myc-TRIM25 and HA-RIPK3 for 24 h and then treated with MG132 (10 mM), rapamycin (100 nM), or Baf A1 (100 nM) for 6 h, and cell lysates were detected by Western blot (left panel). Relative intensity related to internal control GAPDH was analyzed using Image J plugin and plotted by GraphPad Prism 9 (right panel). Error bars indicate the mean (± SD) of three independent experiments. ns, P ≥ 0.05 and ****, P < 0.0001 (one-way ANOVA). ( B ) PK-15 cells infected with CSFV (MOI = 1) were co-transfected with Myc-TRIM25, Flag-RIPK3, and His-Ub for 24 h and then treated with MG132 (10 mM) for 6 h, and the cells were lysed and used for IP experiments. ( C ) HA-RIPK3 and GFP-LC3 were co-transfected into PK-15 cells for 24 h and then treated with rapamycin (100 nM) for 6 h while setting up a control group of cells treated with dimethyl sulfoxide (DMSO). The cells were incubated with anti-HA primary antibody, followed by Alexa fluor 555-conjugated anti-Rabbit-IgG secondary antibody (red), and the nuclei were stained with DAPI (left panel). Scale bars: 100 and 25 µm. The fluorescence intensity profiles of GFP-LC3 (green) and HA-RIPK3 (red) were analyzed using Image J plugin and plotted by GraphPad Prism 9 (right panel). ( D ) PK-15 cells were co-transfected with HA-RIPK3 together with Si TRIM25 for 24 h while setting up a control group of co-transfected Si NC cells, the cells were immunostained with Mito-Tracker, incubated with anti-HA primary antibody, followed by Alexa fluor 488-conjugated anti-Mouse-IgG secondary antibody (green), and nuclei were stained with DAPI (left panel). Scale bars: 100 and 10 µm. The fluorescence intensity profiles of HA-RIPK3 (green) and Mito-Tracker (red) were analyzed using Image J plugin and plotted by GraphPad Prism 9 (middle panel). Pearson’s correlation and overlap co-efficient of HA-RIPK3 and Mito-Tracker were analyzed using Image J plugin and plotted by GraphPad Prism 9 (right panel). Error bars indicate the mean (± SD) of at least three independent experiments. ns, P ≥ 0.05 and *, P < 0.1 (one-way ANOVA). ( E ) PK-15 cells infected with CSFV (MOI = 1) were co-transfected with Si TRIM25, Flag-RIPK3, and GFP-LC3 for 24 h while setting up a control group of co-transfected Si NC cells, the fixed cells were incubated with anti-Flag primary antibody and anti-CD63 primary antibody, respectively, and then stained with Alexa fluor 555-conjugated anti-Mouse-IgG secondary antibody (red) and Dylight 405-labeled anti-Rabbit-IgG secondary antibody (blue) (left panel). Scale bars: 10 µm. The fluorescence intensity profiles of Flag-RIPK3 (blue), GFP-LC3(green), and CD63(red) were analyzed using Image J plugin and plotted by GraphPad Prism 9 (middle panel). Pearson’s correlation and overlap co-efficient were analyzed using Image J plugin and plotted by GraphPad Prism 9 (right panel). Error bars indicate the mean (± SD) of at least three independent experiments. ***, P < 0.001 and ****, P < 0.0001 (one-way ANOVA). ( F ) The mRNA expressions of TRIM25 in the tonsil, lung, lymph node, spleen, kidney, and thymus of CSFV-infected and non-infected piglets were determined by qRT-PCR. The fold changes were related to the internal control β-actin. Error bars indicate the mean (± SD) of three independent experiments. ****, P < 0.0001 (two-way ANOVA). ( G ) To establish PK-15 (upper panel) and 3D4/21 (lower panel) cell models of CSFV infection in vitro , cells were collected at 24 and 48 h post-infection, and protein expression levels of TRIM25 and CSFV N pro were detected by Western blot. ( H ) PK-15 (left panel) and 3D4/21 (right panel) cells infected with CSFV (MOI = 1) were transfected with Myc-TRIM25 or Si TRIM25 for 48 h, and pCMV-N1-Myc or Si NC cells were transfected with a control group. The cell supernatants were collected for TCID 50 assay. Error bars indicate the mean (± SD) of three independent experiments. *, P < 0.1; ***, P < 0.001; ****, P < 0.0001 (one-way ANOVA). ( I ) PK-15 (left panel) and 3D4/21 (right panel) cells infected with CSFV (MOI = 1) were transfected with Myc-TRIM25 for 24 and 48 h, and pCMV-N1-Myc cells were transfected with a control group. Expression levels of TRIM25 and CSFV E2/N pro in cell lysis products were detected by Western blot.

    Techniques Used: Infection, Transfection, Western Blot, Incubation, Staining, Fluorescence, Labeling, Quantitative RT-PCR, In Vitro, Expressing, Lysis

    The autophagy receptor NDP52 binds to RIPK3 and promotes its autophagic degradation. ( A ) HEK-293T cells were co-transfected with HA-RIPK3 together with pCMV-3× Flag, Flag-P62, Flag-NDP52, Flag-NBR1, or Flag-TAX1BP1 for 24 h, the cells were lysed and used for Co-IP experiments, and cell lysates were detected by Western blot. ( B ) Flag-NDP52 and HA-RIPK3 were co-transfected into PK-15 cells for 24 h while setting up a control group of cells transfected with pCMV-3× Flag. The fixed cells were incubated with anti-Flag primary antibody and anti-HA primary antibody, followed by Alexa fluor 488-conjugated anti-Mouse-IgG secondary antibody (green) and Alexa fluor 555-conjugated anti-Rabbit-IgG secondary antibody (red), and the nuclei were stained with DAPI. Scale bars: 100 and 10 µm. ( C ) PK-15 cells infected with CSFV (MOI = 1) were co-transfected with Flag-NDP52, GFP-LC3, and HA-RIPK3 for 24 h and then treated with Baf A1 (100 nM) for 6 h while setting up a control group of cells treated with dimethyl sulfoxide, the fixed cells were incubated with anti-Flag primary antibody and anti-HA primary antibody, respectively, and then stained with Alexa fluor 555-conjugated anti-Rabbit-IgG secondary antibody (red) and Dylight 405-labeled anti-Mouse-IgG secondary antibody (blue). Scale bars: 10 µm. ( D ) Schematic of RIPK3 domain structure and deletion/mutation structure. ( E ) PK-15 cells were co-transfected with Flag-NDP52 together with pCAGGs-HA, Flag-RIPK3 WT, Flag-RIPK3 mutRHIM , Flag-RIPK3 ΔCD, or Flag-RIPK3 ΔKD for 24 h, the cells were lysed and used for Co-IP experiments, and cell lysates were detected by Western blot. ( F ) HEK 293T cells were co-transfected with Flag-NDP52 together with pCAGGs-HA, Flag-RIPK3 WT, Flag-RIPK3 mutRHIM , Flag-RIPK3 ΔCD, or Flag-RIPK3 ΔKD for 24 h, the fixed cells were incubated with anti-Flag primary antibody and anti-HA primary antibody, followed by Alexa fluor 488-conjugated anti-Mouse-IgG secondary antibody (green) and Alexa fluor 555-conjugated anti-Rabbit-IgG secondary antibody (red), and the nuclei were stained with DAPI. Scale bars: 100 and 10 µm. ( G ) Proposed a model by which CSFV infection in vitro limits necroptosis via the autophagy pathway. In vitro , the induction of autophagy by CSFV at a later stage of infection clearly restricts necroptosis. Mechanistic studies revealed that CSFV NS4A protein promoted TRIM25 expression, synergistically induced the occurrence of mitophagy, targeted the autophagic degradation of RIPK3 to block the progression of necroptosis occurrence, and achieved persistent viral infection. Interestingly, we found that RIPK3 was able to specifically localize at the outer mitochondrial membrane, and the autophagy receptor NDP52 was most likely involved in the autophagic degradation of RIPK3 during CSFV infection.
    Figure Legend Snippet: The autophagy receptor NDP52 binds to RIPK3 and promotes its autophagic degradation. ( A ) HEK-293T cells were co-transfected with HA-RIPK3 together with pCMV-3× Flag, Flag-P62, Flag-NDP52, Flag-NBR1, or Flag-TAX1BP1 for 24 h, the cells were lysed and used for Co-IP experiments, and cell lysates were detected by Western blot. ( B ) Flag-NDP52 and HA-RIPK3 were co-transfected into PK-15 cells for 24 h while setting up a control group of cells transfected with pCMV-3× Flag. The fixed cells were incubated with anti-Flag primary antibody and anti-HA primary antibody, followed by Alexa fluor 488-conjugated anti-Mouse-IgG secondary antibody (green) and Alexa fluor 555-conjugated anti-Rabbit-IgG secondary antibody (red), and the nuclei were stained with DAPI. Scale bars: 100 and 10 µm. ( C ) PK-15 cells infected with CSFV (MOI = 1) were co-transfected with Flag-NDP52, GFP-LC3, and HA-RIPK3 for 24 h and then treated with Baf A1 (100 nM) for 6 h while setting up a control group of cells treated with dimethyl sulfoxide, the fixed cells were incubated with anti-Flag primary antibody and anti-HA primary antibody, respectively, and then stained with Alexa fluor 555-conjugated anti-Rabbit-IgG secondary antibody (red) and Dylight 405-labeled anti-Mouse-IgG secondary antibody (blue). Scale bars: 10 µm. ( D ) Schematic of RIPK3 domain structure and deletion/mutation structure. ( E ) PK-15 cells were co-transfected with Flag-NDP52 together with pCAGGs-HA, Flag-RIPK3 WT, Flag-RIPK3 mutRHIM , Flag-RIPK3 ΔCD, or Flag-RIPK3 ΔKD for 24 h, the cells were lysed and used for Co-IP experiments, and cell lysates were detected by Western blot. ( F ) HEK 293T cells were co-transfected with Flag-NDP52 together with pCAGGs-HA, Flag-RIPK3 WT, Flag-RIPK3 mutRHIM , Flag-RIPK3 ΔCD, or Flag-RIPK3 ΔKD for 24 h, the fixed cells were incubated with anti-Flag primary antibody and anti-HA primary antibody, followed by Alexa fluor 488-conjugated anti-Mouse-IgG secondary antibody (green) and Alexa fluor 555-conjugated anti-Rabbit-IgG secondary antibody (red), and the nuclei were stained with DAPI. Scale bars: 100 and 10 µm. ( G ) Proposed a model by which CSFV infection in vitro limits necroptosis via the autophagy pathway. In vitro , the induction of autophagy by CSFV at a later stage of infection clearly restricts necroptosis. Mechanistic studies revealed that CSFV NS4A protein promoted TRIM25 expression, synergistically induced the occurrence of mitophagy, targeted the autophagic degradation of RIPK3 to block the progression of necroptosis occurrence, and achieved persistent viral infection. Interestingly, we found that RIPK3 was able to specifically localize at the outer mitochondrial membrane, and the autophagy receptor NDP52 was most likely involved in the autophagic degradation of RIPK3 during CSFV infection.

    Techniques Used: Transfection, Co-Immunoprecipitation Assay, Western Blot, Incubation, Staining, Infection, Labeling, Mutagenesis, In Vitro, Expressing, Blocking Assay, Membrane

    swine kidney cell line pk 15  (ATCC)


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    ATCC swine kidney cell line pk 15
    Swine Kidney Cell Line Pk 15, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC swine kidney cell line pk 15
    FMDV infection-induced autophagy plays an important role in viral replication. ( A ) <t>PK-15</t> cells were mock infected or infected with FMDV (MOI = 1) for 0, 0.5, 1, 1.5, 2 and 3 h. The expression of LC3B and SQSTM1 was analyzed by western blot. The level of 3D was analyzed by RT-PCR. ACTB and GAPDH were used as a sample loading control. ( B ) The cells were then fixed and processed for indirect immunofluorescence using antibodies against LC3B and the 3D protein, followed by the corresponding secondary antibodies. The fluorescence signals were visualized by confocal immunofluorescence microscopy. ( C ) ATG5 knockdown (KD) and wild-type cells were infected with FMDV (MOI = 1) for 2 and 3 h. The expression of ATG5 and LC3B was analyzed by western blot. ( D and E ) ATG5 KD and wild-type cells were infected with FMDV (MOI = 1). At 3 hpi, both the extracellular and intracellular copy numbers of FMDV were measured by qRT-PCR; both the extracellular and intracellular virus titers were measured by TCID 50 . The data represent the mean ± SD of 3 independent experiments. ***P < 0.001. ( F ) PK-15 cells were mock infected or infected with FMDV (MOI = 1) for 6 and 9 h. The expression of SQSTM1 and VP1 were analyzed by western blot. ( G ) Cells were transfected with pmCherry-GFP-LC3B for 24 h, followed by FMDV infection (MOI = 1) and treatment with Baf-A1. The fluorescence signals were visualized by confocal immunofluorescence microscopy. Scale bars: 10 μm.
    Swine Kidney Cell Line Pk 15, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Foot-and-mouth disease virus capsid protein VP2 activates the cellular EIF2S1-ATF4 pathway and induces autophagy via HSPB1"

    Article Title: Foot-and-mouth disease virus capsid protein VP2 activates the cellular EIF2S1-ATF4 pathway and induces autophagy via HSPB1

    Journal: Autophagy

    doi: 10.1080/15548627.2017.1405187

    FMDV infection-induced autophagy plays an important role in viral replication. ( A ) PK-15 cells were mock infected or infected with FMDV (MOI = 1) for 0, 0.5, 1, 1.5, 2 and 3 h. The expression of LC3B and SQSTM1 was analyzed by western blot. The level of 3D was analyzed by RT-PCR. ACTB and GAPDH were used as a sample loading control. ( B ) The cells were then fixed and processed for indirect immunofluorescence using antibodies against LC3B and the 3D protein, followed by the corresponding secondary antibodies. The fluorescence signals were visualized by confocal immunofluorescence microscopy. ( C ) ATG5 knockdown (KD) and wild-type cells were infected with FMDV (MOI = 1) for 2 and 3 h. The expression of ATG5 and LC3B was analyzed by western blot. ( D and E ) ATG5 KD and wild-type cells were infected with FMDV (MOI = 1). At 3 hpi, both the extracellular and intracellular copy numbers of FMDV were measured by qRT-PCR; both the extracellular and intracellular virus titers were measured by TCID 50 . The data represent the mean ± SD of 3 independent experiments. ***P < 0.001. ( F ) PK-15 cells were mock infected or infected with FMDV (MOI = 1) for 6 and 9 h. The expression of SQSTM1 and VP1 were analyzed by western blot. ( G ) Cells were transfected with pmCherry-GFP-LC3B for 24 h, followed by FMDV infection (MOI = 1) and treatment with Baf-A1. The fluorescence signals were visualized by confocal immunofluorescence microscopy. Scale bars: 10 μm.
    Figure Legend Snippet: FMDV infection-induced autophagy plays an important role in viral replication. ( A ) PK-15 cells were mock infected or infected with FMDV (MOI = 1) for 0, 0.5, 1, 1.5, 2 and 3 h. The expression of LC3B and SQSTM1 was analyzed by western blot. The level of 3D was analyzed by RT-PCR. ACTB and GAPDH were used as a sample loading control. ( B ) The cells were then fixed and processed for indirect immunofluorescence using antibodies against LC3B and the 3D protein, followed by the corresponding secondary antibodies. The fluorescence signals were visualized by confocal immunofluorescence microscopy. ( C ) ATG5 knockdown (KD) and wild-type cells were infected with FMDV (MOI = 1) for 2 and 3 h. The expression of ATG5 and LC3B was analyzed by western blot. ( D and E ) ATG5 KD and wild-type cells were infected with FMDV (MOI = 1). At 3 hpi, both the extracellular and intracellular copy numbers of FMDV were measured by qRT-PCR; both the extracellular and intracellular virus titers were measured by TCID 50 . The data represent the mean ± SD of 3 independent experiments. ***P < 0.001. ( F ) PK-15 cells were mock infected or infected with FMDV (MOI = 1) for 6 and 9 h. The expression of SQSTM1 and VP1 were analyzed by western blot. ( G ) Cells were transfected with pmCherry-GFP-LC3B for 24 h, followed by FMDV infection (MOI = 1) and treatment with Baf-A1. The fluorescence signals were visualized by confocal immunofluorescence microscopy. Scale bars: 10 μm.

    Techniques Used: Infection, Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Fluorescence, Microscopy, Quantitative RT-PCR, Transfection

    FMDV infection induces autophagy through the EIF2S1-ATF4-AKT-MTOR cascade. ( A ) PK-15 cells were mock infected or infected with FMDV (MOI = 1) for 1.5, 2 and 3 h. The phosphorylation of AKT, AMPK, MTOR and ULK1 were analyzed by western blot. ACTB was used as a sample loading control. ( B ) PK-15 cells were mock infected or infected with FMDV (MOI = 1) for 1.5, 2 and 3 h. ATF4 and phosphorylation of EIF2S1 were analyzed by western blot. ( C ) ATF4 KD and wild-type cells cells were infected with FMDV (MOI = 1). ATF4, LC3B and phosphorylation of AKT, MTOR and ULK1 were analyzed by western blot. ( D ) ATF4 and scrambled knockdown cells were transfected with pmCherry-GFP-LC3B for 24 h, followed by FMDV infection (MOI = 1) for 3 h. The fluorescence signals were visualized by confocal immunofluorescence microscopy. Scale bars: 10 μm. ( E and F ) ATF4 KD and wil-type cells cells were infected with FMDV (MOI = 1) for 3 h. At 3 hpi, both the extracellular and intracellular copy numbers of FMDV were measured by qRT-PCR; both the extracellular and intracellular virus titers were measured by TCID 50 . The data represent the mean ± SD of 3 independent experiments. **P < 0.01, ***P < 0.001.
    Figure Legend Snippet: FMDV infection induces autophagy through the EIF2S1-ATF4-AKT-MTOR cascade. ( A ) PK-15 cells were mock infected or infected with FMDV (MOI = 1) for 1.5, 2 and 3 h. The phosphorylation of AKT, AMPK, MTOR and ULK1 were analyzed by western blot. ACTB was used as a sample loading control. ( B ) PK-15 cells were mock infected or infected with FMDV (MOI = 1) for 1.5, 2 and 3 h. ATF4 and phosphorylation of EIF2S1 were analyzed by western blot. ( C ) ATF4 KD and wild-type cells cells were infected with FMDV (MOI = 1). ATF4, LC3B and phosphorylation of AKT, MTOR and ULK1 were analyzed by western blot. ( D ) ATF4 and scrambled knockdown cells were transfected with pmCherry-GFP-LC3B for 24 h, followed by FMDV infection (MOI = 1) for 3 h. The fluorescence signals were visualized by confocal immunofluorescence microscopy. Scale bars: 10 μm. ( E and F ) ATF4 KD and wil-type cells cells were infected with FMDV (MOI = 1) for 3 h. At 3 hpi, both the extracellular and intracellular copy numbers of FMDV were measured by qRT-PCR; both the extracellular and intracellular virus titers were measured by TCID 50 . The data represent the mean ± SD of 3 independent experiments. **P < 0.01, ***P < 0.001.

    Techniques Used: Infection, Western Blot, Transfection, Fluorescence, Immunofluorescence, Microscopy, Quantitative RT-PCR

    UV-FMDV infection induces autophagy. ( A ) PK-15 cells were mock infected or infected with UV-FMDV for 3 h (MOI = 10). LC3B and phosphorylation of EIF2S1, AKT and MTOR were analyzed by western blot. ACTB was used as a sample loading control. ( B ) ATF4 KD and wild-type cells were infected as described in ( A ).
    Figure Legend Snippet: UV-FMDV infection induces autophagy. ( A ) PK-15 cells were mock infected or infected with UV-FMDV for 3 h (MOI = 10). LC3B and phosphorylation of EIF2S1, AKT and MTOR were analyzed by western blot. ACTB was used as a sample loading control. ( B ) ATF4 KD and wild-type cells were infected as described in ( A ).

    Techniques Used: Infection, Western Blot

    FMDV capsid protein VP2 induced autophagy. ( A ) PK-15 cells were transfected with empty vectors or various plasmids expressing FLAG-tagged VP1, VP2and VP3 proteins for 24 h. LC3B and FMDV capsid proteins were analyzed by western blot. ACTB was used as a sample loading control. ( B ) Cells were transfected with empty vectors or pCMV-Flag-VP2 for 24 h. Cells were fixed and analyzed by immunofluorescence using anti-LC3B antibodies. The fluorescence signals were visualized by confocal immunofluorescence microscopy. Scale bars: 10 μm. ( C ) PK-15 cells were transfected with empty vectors or pCMV-Flag-VP2 for 24 h. LC3B and phosphorylation of EIF2S1, AKT and MTOR were analyzed by western blot. ACTB was used as a sample loading control. ( D ) ATF4 and scrambled knockdown cells were transfected with pCMV-Flag-VP2 for 24 h. LC3B and phosphorylation of AKT and MTOR were analyzed by western blot. ACTB was used as a sample loading control.
    Figure Legend Snippet: FMDV capsid protein VP2 induced autophagy. ( A ) PK-15 cells were transfected with empty vectors or various plasmids expressing FLAG-tagged VP1, VP2and VP3 proteins for 24 h. LC3B and FMDV capsid proteins were analyzed by western blot. ACTB was used as a sample loading control. ( B ) Cells were transfected with empty vectors or pCMV-Flag-VP2 for 24 h. Cells were fixed and analyzed by immunofluorescence using anti-LC3B antibodies. The fluorescence signals were visualized by confocal immunofluorescence microscopy. Scale bars: 10 μm. ( C ) PK-15 cells were transfected with empty vectors or pCMV-Flag-VP2 for 24 h. LC3B and phosphorylation of EIF2S1, AKT and MTOR were analyzed by western blot. ACTB was used as a sample loading control. ( D ) ATF4 and scrambled knockdown cells were transfected with pCMV-Flag-VP2 for 24 h. LC3B and phosphorylation of AKT and MTOR were analyzed by western blot. ACTB was used as a sample loading control.

    Techniques Used: Transfection, Expressing, Western Blot, Immunofluorescence, Fluorescence, Microscopy

    VP2 mutant and interaction between VP2 and HSPB1 in PK-15 cells. ( A ) A scheme of the VP2 muant. ( B ) PK-15 cells were transfected with empty vectors, pCMV-Flag-VP2, or VP2 mutant for 24 h. LC3B and ACTB were analyzed by western blot. ( C and D ) The interaction between FMDV VP2 and HSPB1 in PK-15 cells was verified. The PK-15 cells were co-transfected with 10 μg pCMV-Flag-VP2 plasmid and 10 μg pEGFP-HA-HSPB1 plasmid or transfected with just 10 μg pCMV-Flag-VP2 plasmid, and immunoprecipitation was performed with anti-HA antibody. Immunoblotting analysis was performed with anti-HA antibody and anti-FLAG antibody. ( E and F ) The PK-15 cells were transfected with 10 μg pCMV-Flag-VP2 plasmid or 10 μg empty vector pCMV-Flag plasmid, and immunoprecipitation was performed with anti-FLAG antibody. Immunoblotting analysis was performed with anti-FLAG antibody and anti-HSPB1 antibody.
    Figure Legend Snippet: VP2 mutant and interaction between VP2 and HSPB1 in PK-15 cells. ( A ) A scheme of the VP2 muant. ( B ) PK-15 cells were transfected with empty vectors, pCMV-Flag-VP2, or VP2 mutant for 24 h. LC3B and ACTB were analyzed by western blot. ( C and D ) The interaction between FMDV VP2 and HSPB1 in PK-15 cells was verified. The PK-15 cells were co-transfected with 10 μg pCMV-Flag-VP2 plasmid and 10 μg pEGFP-HA-HSPB1 plasmid or transfected with just 10 μg pCMV-Flag-VP2 plasmid, and immunoprecipitation was performed with anti-HA antibody. Immunoblotting analysis was performed with anti-HA antibody and anti-FLAG antibody. ( E and F ) The PK-15 cells were transfected with 10 μg pCMV-Flag-VP2 plasmid or 10 μg empty vector pCMV-Flag plasmid, and immunoprecipitation was performed with anti-FLAG antibody. Immunoblotting analysis was performed with anti-FLAG antibody and anti-HSPB1 antibody.

    Techniques Used: Mutagenesis, Transfection, Western Blot, Plasmid Preparation, Immunoprecipitation

    VP2 decreases aggregation of HTT103Q. ( A ) HEK293T cells and ( B) Vero cells were transfected with empty vectors or pCMV-N-VP2 for 24 h. LC3B and SQSTM1 by were analyzed by western blot. ACTB was used as a sample loading control. ( C ) PK-15 cells were transfected with empty vectors or pCMV-EGFP-HTT103Q for 24 h. HTT103Q and SQSTM1 were analyzed by western blot. ACTB was used as a sample loading control. ( D ) PK-15 cells and ATG5 KD cells were co-transfected with pCMV-Flag-VP2 and pCMV-EGFP-HTT103Q for 24 h. The fluorescence signals were visualized by confocal immunofluorescence microscopy. Scale bars: 10 μm. ( E ) The number of HTT103Q dot was counted. The data represent the mean ± SD of 3 independent experiments. **P < 0.01.
    Figure Legend Snippet: VP2 decreases aggregation of HTT103Q. ( A ) HEK293T cells and ( B) Vero cells were transfected with empty vectors or pCMV-N-VP2 for 24 h. LC3B and SQSTM1 by were analyzed by western blot. ACTB was used as a sample loading control. ( C ) PK-15 cells were transfected with empty vectors or pCMV-EGFP-HTT103Q for 24 h. HTT103Q and SQSTM1 were analyzed by western blot. ACTB was used as a sample loading control. ( D ) PK-15 cells and ATG5 KD cells were co-transfected with pCMV-Flag-VP2 and pCMV-EGFP-HTT103Q for 24 h. The fluorescence signals were visualized by confocal immunofluorescence microscopy. Scale bars: 10 μm. ( E ) The number of HTT103Q dot was counted. The data represent the mean ± SD of 3 independent experiments. **P < 0.01.

    Techniques Used: Transfection, Western Blot, Fluorescence, Immunofluorescence, Microscopy

    swine kidney cell line pk 15  (ATCC)


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    ATCC swine kidney cell line pk 15
    CSFV infection Up-regulates HSP90AA1 expression. (A) HSP90AA1 mRNA relative levels in <t>PK-15</t> and 3D4/2 cells was analyzed by qRT-PCR; (B) Western blot showing HSP90AA1 protein expression and the relative protein levels of HSP90AA1 in PK-15 and 3D4/2 cells were estimated by histograms representing density readings of the gel bands with Image J, and the ratios were calculated relative to Tubulin control. (*p < 0.05, **p < 0.01 and ****p < 0.0001 calculated using two-way ANOVA, ns, not significant).
    Swine Kidney Cell Line Pk 15, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "HSP90AA1 interacts with CSFV NS5A protein and regulates CSFV replication via the JAK/STAT and NF-κB signaling pathway"

    Article Title: HSP90AA1 interacts with CSFV NS5A protein and regulates CSFV replication via the JAK/STAT and NF-κB signaling pathway

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2022.1031868

    CSFV infection Up-regulates HSP90AA1 expression. (A) HSP90AA1 mRNA relative levels in PK-15 and 3D4/2 cells was analyzed by qRT-PCR; (B) Western blot showing HSP90AA1 protein expression and the relative protein levels of HSP90AA1 in PK-15 and 3D4/2 cells were estimated by histograms representing density readings of the gel bands with Image J, and the ratios were calculated relative to Tubulin control. (*p < 0.05, **p < 0.01 and ****p < 0.0001 calculated using two-way ANOVA, ns, not significant).
    Figure Legend Snippet: CSFV infection Up-regulates HSP90AA1 expression. (A) HSP90AA1 mRNA relative levels in PK-15 and 3D4/2 cells was analyzed by qRT-PCR; (B) Western blot showing HSP90AA1 protein expression and the relative protein levels of HSP90AA1 in PK-15 and 3D4/2 cells were estimated by histograms representing density readings of the gel bands with Image J, and the ratios were calculated relative to Tubulin control. (*p < 0.05, **p < 0.01 and ****p < 0.0001 calculated using two-way ANOVA, ns, not significant).

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

    Over-expression of HSP90AA1 inhibits CSFV replication. (A) CSFV NS5B copy numbers were determined by RT-PCR at 24 and 48 hpi in HSP90AA1-overexpression PK-15 and 3D4/2 cells. (B) Western blot for HSP90AA1 and CSFV N pro expression in HSP90AA1-overexpression PK-15 and 3D4/2 cells, and the relative protein levels of N pro in HSP90AA1-overexpression PK-15 and 3D4/2 cells were estimated by histograms representing density readings of the gel bands with Image J, and the ratios were calculated relative to Tubulin control. (C) Infectious progeny viral titers in supernatants from HSP90AA1-overexpressing PK-15 and 3D4/2 cells. Viral titers from the supernatants collected at 24 and 48 hpi were determined and expressed as TCID 50 /ml. (*p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 calculated using two-way ANOVA. ns, not significant).
    Figure Legend Snippet: Over-expression of HSP90AA1 inhibits CSFV replication. (A) CSFV NS5B copy numbers were determined by RT-PCR at 24 and 48 hpi in HSP90AA1-overexpression PK-15 and 3D4/2 cells. (B) Western blot for HSP90AA1 and CSFV N pro expression in HSP90AA1-overexpression PK-15 and 3D4/2 cells, and the relative protein levels of N pro in HSP90AA1-overexpression PK-15 and 3D4/2 cells were estimated by histograms representing density readings of the gel bands with Image J, and the ratios were calculated relative to Tubulin control. (C) Infectious progeny viral titers in supernatants from HSP90AA1-overexpressing PK-15 and 3D4/2 cells. Viral titers from the supernatants collected at 24 and 48 hpi were determined and expressed as TCID 50 /ml. (*p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 calculated using two-way ANOVA. ns, not significant).

    Techniques Used: Over Expression, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing

    Knockdown of HSP90AA1 promoted CSFV replication. (A) RT-PCR determined CSFV NS5B copy numbers at 24 and 48 hpi in HSP90AA1 knockdown PK-15 and 3D4/2 cells. (B) Western blot for HSP90AA1 and CSFV N pro expression in HSP90AA1 knock-downed PK-15 and 3D4/2 cells, and the relative levels of N pro in HSP90AA1 knock-downed PK-15 and 3D4/2 cells were estimated by histograms representing density readings of the gel bands with Image J, and the ratios were calculated relative to tubulin control. (C) Infectious progeny viral titers in supernatants from HSP90AA1-knockdown PK-15 and 3D4/2 cells. Viral titers from the supernatant collected at 24 and 48 hpi were determined and expressed as TCID 50 /ml. (*p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 calculated using two-way ANOVA, ns, not significant).
    Figure Legend Snippet: Knockdown of HSP90AA1 promoted CSFV replication. (A) RT-PCR determined CSFV NS5B copy numbers at 24 and 48 hpi in HSP90AA1 knockdown PK-15 and 3D4/2 cells. (B) Western blot for HSP90AA1 and CSFV N pro expression in HSP90AA1 knock-downed PK-15 and 3D4/2 cells, and the relative levels of N pro in HSP90AA1 knock-downed PK-15 and 3D4/2 cells were estimated by histograms representing density readings of the gel bands with Image J, and the ratios were calculated relative to tubulin control. (C) Infectious progeny viral titers in supernatants from HSP90AA1-knockdown PK-15 and 3D4/2 cells. Viral titers from the supernatant collected at 24 and 48 hpi were determined and expressed as TCID 50 /ml. (*p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 calculated using two-way ANOVA, ns, not significant).

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing

    Validation for the interaction of HSP90AA1 with CSFV NS5A protein. (A) Identification of the colocalization of HSP90AA1 with NS5A protein in 293T cells. 293T cells co-expressing pEGFP-NS5A and p3×Flag-HSP90AA1 were analyzed by laser-scanning confocal microscopy. 293T Cells were co-transfected with pEGFP and p3×Flag-HSP90AA1 and pEGFP-NS5A with p3×Flag-CMV as negative controls. (B) Exogenous Co-IP analysis of NS5A with HSP90AA1 in 293T cells. 293T cells were co-transfected with p3×Flag-HSP90AA1 and EGFP-NS5A. 293T Cells were co-transfected with pEGFP and p3×Flag-HSP90AA1 and pEGFP-NS5A with p3×Flag-CMV as negative controls. (C, D) Identification of the colocalization of HSP90AA1 with NS5A protein in PK and 3D4/2 cells. PK-15 and 3D4/2 cells co-expressing pEGFP-NS5A and p3×Flag-HSP90AA1 were analyzed by laser-scanning confocal microscopy. PK-15 and 3D4/2 Cells were co-transfected with pEGFP and p3×Flag-HSP90AA1 and pEGFP-NS5A with p3×Flag-CMV as negative controls.
    Figure Legend Snippet: Validation for the interaction of HSP90AA1 with CSFV NS5A protein. (A) Identification of the colocalization of HSP90AA1 with NS5A protein in 293T cells. 293T cells co-expressing pEGFP-NS5A and p3×Flag-HSP90AA1 were analyzed by laser-scanning confocal microscopy. 293T Cells were co-transfected with pEGFP and p3×Flag-HSP90AA1 and pEGFP-NS5A with p3×Flag-CMV as negative controls. (B) Exogenous Co-IP analysis of NS5A with HSP90AA1 in 293T cells. 293T cells were co-transfected with p3×Flag-HSP90AA1 and EGFP-NS5A. 293T Cells were co-transfected with pEGFP and p3×Flag-HSP90AA1 and pEGFP-NS5A with p3×Flag-CMV as negative controls. (C, D) Identification of the colocalization of HSP90AA1 with NS5A protein in PK and 3D4/2 cells. PK-15 and 3D4/2 cells co-expressing pEGFP-NS5A and p3×Flag-HSP90AA1 were analyzed by laser-scanning confocal microscopy. PK-15 and 3D4/2 Cells were co-transfected with pEGFP and p3×Flag-HSP90AA1 and pEGFP-NS5A with p3×Flag-CMV as negative controls.

    Techniques Used: Expressing, Confocal Microscopy, Transfection, Co-Immunoprecipitation Assay

    HSP90AA1 reduces the protein levels of CSFV NS5A. (A, B) EGFP-NS5A or EGFP-NS4A protein expression in HSP90AA1-expressing PK-15 and 3D4/2 cells. PK-15 and 3D4/2 cells were co-transfected with pEGFP-NS5A or EGFP-NS4A with a different amount of p3×Flag-HSP90AA1. Western blot analyzed EGFP-NS5A or EGFP-NS4A protein expression at 24 hours post co-transfected. Tubulin served as an internal control. The relative levels of EGFP-NS5A or EGFP-NS4A were estimated by histograms representing density reading of the gel bands with Image J, and the ratios were calculated relative to tubulin control. (*p < 0.05, **p < 0.01 and ***p < 0.001 calculated using one-way ANOVA, ns, not significant).
    Figure Legend Snippet: HSP90AA1 reduces the protein levels of CSFV NS5A. (A, B) EGFP-NS5A or EGFP-NS4A protein expression in HSP90AA1-expressing PK-15 and 3D4/2 cells. PK-15 and 3D4/2 cells were co-transfected with pEGFP-NS5A or EGFP-NS4A with a different amount of p3×Flag-HSP90AA1. Western blot analyzed EGFP-NS5A or EGFP-NS4A protein expression at 24 hours post co-transfected. Tubulin served as an internal control. The relative levels of EGFP-NS5A or EGFP-NS4A were estimated by histograms representing density reading of the gel bands with Image J, and the ratios were calculated relative to tubulin control. (*p < 0.05, **p < 0.01 and ***p < 0.001 calculated using one-way ANOVA, ns, not significant).

    Techniques Used: Expressing, Transfection, Western Blot

    HSP90AA1 over-expression activates STAT1 and P65 in PK-15 and 3D4/2 cells. (A) HSP90AA1 over-expression affected the phosphorylation of STAT1 and nuclear translocation of p-STAT1 in PK-15 and 3D4/2 cells were observed by laser confocal microscopy. (B) HSP90AA1 over-expression affected the nuclear translocation of p-P65 in PK-15 and 3D4/2 cells were observed by laser confocal microscopy.
    Figure Legend Snippet: HSP90AA1 over-expression activates STAT1 and P65 in PK-15 and 3D4/2 cells. (A) HSP90AA1 over-expression affected the phosphorylation of STAT1 and nuclear translocation of p-STAT1 in PK-15 and 3D4/2 cells were observed by laser confocal microscopy. (B) HSP90AA1 over-expression affected the nuclear translocation of p-P65 in PK-15 and 3D4/2 cells were observed by laser confocal microscopy.

    Techniques Used: Over Expression, Translocation Assay, Confocal Microscopy

    HSP90AA1 over-expression does not promote the expressions of ISGs and IFN-α under CSFV Infection. (A, B) Relative mRNA levels of ISGs and IFN-α in PK-15 and 3D4/2 cells were determined by RT-PCR. Cells were transfected with p3×Flag-HSP90AA1 first and then infected with CSFV (MOI=1). Cells were harvested at 24hpi and 48hpi. Total cellular RNA was extracted to determine relative mRNA expression levels of ISGs and IFN-α. (*p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 calculated using two-way ANOVA, ns, not significant).
    Figure Legend Snippet: HSP90AA1 over-expression does not promote the expressions of ISGs and IFN-α under CSFV Infection. (A, B) Relative mRNA levels of ISGs and IFN-α in PK-15 and 3D4/2 cells were determined by RT-PCR. Cells were transfected with p3×Flag-HSP90AA1 first and then infected with CSFV (MOI=1). Cells were harvested at 24hpi and 48hpi. Total cellular RNA was extracted to determine relative mRNA expression levels of ISGs and IFN-α. (*p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 calculated using two-way ANOVA, ns, not significant).

    Techniques Used: Over Expression, Infection, Reverse Transcription Polymerase Chain Reaction, Transfection, Expressing

    CSFV infection antagonizes the activation of HSP90AA1 on JAK/STAT pathway. (A, B) Western blot for JAK1, STAT1, p-JAK1, p-STAT1, OAS2, ISG-15 and HSP90AA1 expression in HSP90AA1-overexpression or knock-downed PK-15 and 3D4/2 cells. Cells were infected with CSFV (MOI=1) after transfection p3×Flag-HSP90AA1 or siHSP90AA1. The cells were not infected with CSFV as a control. Cells were harvested at 24hpi and 48hpi served to Western blot. The relative levels of proteins were estimated by histograms representing density reading of the gel bands with Image J, and the ratios were calculated relative to tubulin control. (*p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 calculated using two-way ANOVA, ns, not significant).
    Figure Legend Snippet: CSFV infection antagonizes the activation of HSP90AA1 on JAK/STAT pathway. (A, B) Western blot for JAK1, STAT1, p-JAK1, p-STAT1, OAS2, ISG-15 and HSP90AA1 expression in HSP90AA1-overexpression or knock-downed PK-15 and 3D4/2 cells. Cells were infected with CSFV (MOI=1) after transfection p3×Flag-HSP90AA1 or siHSP90AA1. The cells were not infected with CSFV as a control. Cells were harvested at 24hpi and 48hpi served to Western blot. The relative levels of proteins were estimated by histograms representing density reading of the gel bands with Image J, and the ratios were calculated relative to tubulin control. (*p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 calculated using two-way ANOVA, ns, not significant).

    Techniques Used: Infection, Activation Assay, Western Blot, Expressing, Over Expression, Transfection

    CSFV infection antagonizes the activation of HSP90AA1 on NF-κB pathway. (A, B) Western blot for P65, p-IκBα and IκBα in HSP90AA1-overexpression or knock-downed PK-15 and 3D4/2 cells. Cells were infected with CSFV (MOI=1) after transfection p3×Flag-HSP90AA1 or siHSP90AA1, and the cells were not infected with CSFV as a control. Cells were harvested at 24hpi and 48hpi served to Western blot. The relative levels of proteins were estimated by histograms representing density reading of the gel bands with Image J, and the ratios were calculated relative to tubulin control. (*p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 calculated using two-way ANOVA, ns, not significant).
    Figure Legend Snippet: CSFV infection antagonizes the activation of HSP90AA1 on NF-κB pathway. (A, B) Western blot for P65, p-IκBα and IκBα in HSP90AA1-overexpression or knock-downed PK-15 and 3D4/2 cells. Cells were infected with CSFV (MOI=1) after transfection p3×Flag-HSP90AA1 or siHSP90AA1, and the cells were not infected with CSFV as a control. Cells were harvested at 24hpi and 48hpi served to Western blot. The relative levels of proteins were estimated by histograms representing density reading of the gel bands with Image J, and the ratios were calculated relative to tubulin control. (*p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 calculated using two-way ANOVA, ns, not significant).

    Techniques Used: Infection, Activation Assay, Western Blot, Over Expression, Transfection

    swine kidney cell line pk 15  (ATCC)


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    ATCC swine kidney cell line pk 15
    Swine Kidney Cell Line Pk 15, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    swine kidney cell line pk 15  (ATCC)


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    ATCC swine kidney cell line pk 15
    PRV induced cell death with necrotic characteristics. (A) PI-positive <t>PK-15</t> cells were increased by PRV infection. PK-15 cells were mock-infected or infected with PRV GD-WH or RA strains (MOI = 10) for 12 and 24 h. After cells were stained with PI (red), live images were snapped. (B) Statistical analysis of the ratio of PI-positive cells in live images (mean ± SD; n = 3; ** P < 0.01, *** P < 0.001). P values were calculated by using two-way ANOVA. (C) Cell viability was decreased by PRV infection. PK-15 cells were mock-infected or infected with PRV GD-WH or RA strains (MOI = 10) for 24 h. Cell viability was analyzed by using the CCK8 assay as described in the Materials and Methods section (mean ± SD; n = 3; ** P < 0.01, ***P < 0.001). P values were calculated by using two-way ANOVA. (D) Disrupted membrane of PRV-infected cells was observed by using TEM. PK-15 cells in 10-cm dishes were mock-infected or PRV-infected at an MOI of 10 for 24 h. Then, cell monolayer was collected and analyzed by using TEM. In TEM images, N indicated the cell nucleus. Arrowheads point to cell plasma membrane leakage. (E) Quantification ratio of PK-15 with the disrupted cell plasma membrane (mean ± SD; n ≥ 30 cells; ***P < 0.001). P values were calculated by using an unpaired Student’s t -test.
    Swine Kidney Cell Line Pk 15, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "RIPK3-Dependent Necroptosis Limits PRV Replication in PK-15 Cells"

    Article Title: RIPK3-Dependent Necroptosis Limits PRV Replication in PK-15 Cells

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2021.664353

    PRV induced cell death with necrotic characteristics. (A) PI-positive PK-15 cells were increased by PRV infection. PK-15 cells were mock-infected or infected with PRV GD-WH or RA strains (MOI = 10) for 12 and 24 h. After cells were stained with PI (red), live images were snapped. (B) Statistical analysis of the ratio of PI-positive cells in live images (mean ± SD; n = 3; ** P < 0.01, *** P < 0.001). P values were calculated by using two-way ANOVA. (C) Cell viability was decreased by PRV infection. PK-15 cells were mock-infected or infected with PRV GD-WH or RA strains (MOI = 10) for 24 h. Cell viability was analyzed by using the CCK8 assay as described in the Materials and Methods section (mean ± SD; n = 3; ** P < 0.01, ***P < 0.001). P values were calculated by using two-way ANOVA. (D) Disrupted membrane of PRV-infected cells was observed by using TEM. PK-15 cells in 10-cm dishes were mock-infected or PRV-infected at an MOI of 10 for 24 h. Then, cell monolayer was collected and analyzed by using TEM. In TEM images, N indicated the cell nucleus. Arrowheads point to cell plasma membrane leakage. (E) Quantification ratio of PK-15 with the disrupted cell plasma membrane (mean ± SD; n ≥ 30 cells; ***P < 0.001). P values were calculated by using an unpaired Student’s t -test.
    Figure Legend Snippet: PRV induced cell death with necrotic characteristics. (A) PI-positive PK-15 cells were increased by PRV infection. PK-15 cells were mock-infected or infected with PRV GD-WH or RA strains (MOI = 10) for 12 and 24 h. After cells were stained with PI (red), live images were snapped. (B) Statistical analysis of the ratio of PI-positive cells in live images (mean ± SD; n = 3; ** P < 0.01, *** P < 0.001). P values were calculated by using two-way ANOVA. (C) Cell viability was decreased by PRV infection. PK-15 cells were mock-infected or infected with PRV GD-WH or RA strains (MOI = 10) for 24 h. Cell viability was analyzed by using the CCK8 assay as described in the Materials and Methods section (mean ± SD; n = 3; ** P < 0.01, ***P < 0.001). P values were calculated by using two-way ANOVA. (D) Disrupted membrane of PRV-infected cells was observed by using TEM. PK-15 cells in 10-cm dishes were mock-infected or PRV-infected at an MOI of 10 for 24 h. Then, cell monolayer was collected and analyzed by using TEM. In TEM images, N indicated the cell nucleus. Arrowheads point to cell plasma membrane leakage. (E) Quantification ratio of PK-15 with the disrupted cell plasma membrane (mean ± SD; n ≥ 30 cells; ***P < 0.001). P values were calculated by using an unpaired Student’s t -test.

    Techniques Used: Infection, Staining, CCK-8 Assay

    Cell death induced by PRV infection was caspase independent. (A) Z-VAD-FMK treatment has no inhibitory effect on increased PI-positive cells during PRV infection. PK-15 cells pretreated with 10 μM Z-VAD-FMK were mock-infected or infected with PRV GD-WH or RA strains (MOI = 10) for 24 h. After cells were stained with Hoechst-33342 (blue) and PI (red), live images were snapped. BF indicated the bright field images. H + P indicated the merged images of cells stained with Hoechst-33342 (blue) and PI (red). (B) Flow cytometry analysis of the effect of Z-VAD-FMK treatment on PRV-infected PK-15 cells. PK-15 cells pretreated with 10 μM Z-VAD-FMK were mock-infected or infected with PRV GD-WH or RA strains (MOI = 10) for 24 h. Rates of apoptotic cells were analyzed by using the Alexa Fluor TM 488 Annexin V/Dead Cell Apoptosis Detection Kit as described in the Materials and Methods section (Q1-UL, Annexin V-negative and PI-positive; Q1-UR, Annexin V-positive and PI-positive; Q1-LL, Annexin V-negative and PI-negative; Q1-LR, Annexin-V-positive and PI-negative). (C) Statistical analysis of the ratio of apoptotic cells (Annexin-V-positive and PI-negative) or dead cells (PI-positive) in flow cytometry analysis data (mean ± SD; n = 3; NS P > 0.05, *** P < 0.001). P values were calculated by using two-way ANOVA. (D) The decline of cell viability induced by PRV infection cannot be recovered by Z-VAD-FMK treatment. PK-15 cells pretreated with 10 μM Z-VAD-FMK were mock-infected or infected with PRV GD-WH or RA strains (MOI = 10) for 24 h. Cell viability was analyzed by using the CCK8 assay as described in the Materials and Methods section (mean ± SD; n = 3; NS P > 0.05, ***P < 0.001). P values were calculated by using an unpaired Student’s t -test. (E) Western blot analysis showed that caspase proteins were not activated during PRV infection. PK-15 cells were mock-infected, infected with PRV GD-WH or RA strains (MOI = 10), or 50 nM staurosporine treated for 24 h. Caspase-3 and caspase-8 in whole cell lysates were analyzed by using Western blot as described in the Materials and Methods section.
    Figure Legend Snippet: Cell death induced by PRV infection was caspase independent. (A) Z-VAD-FMK treatment has no inhibitory effect on increased PI-positive cells during PRV infection. PK-15 cells pretreated with 10 μM Z-VAD-FMK were mock-infected or infected with PRV GD-WH or RA strains (MOI = 10) for 24 h. After cells were stained with Hoechst-33342 (blue) and PI (red), live images were snapped. BF indicated the bright field images. H + P indicated the merged images of cells stained with Hoechst-33342 (blue) and PI (red). (B) Flow cytometry analysis of the effect of Z-VAD-FMK treatment on PRV-infected PK-15 cells. PK-15 cells pretreated with 10 μM Z-VAD-FMK were mock-infected or infected with PRV GD-WH or RA strains (MOI = 10) for 24 h. Rates of apoptotic cells were analyzed by using the Alexa Fluor TM 488 Annexin V/Dead Cell Apoptosis Detection Kit as described in the Materials and Methods section (Q1-UL, Annexin V-negative and PI-positive; Q1-UR, Annexin V-positive and PI-positive; Q1-LL, Annexin V-negative and PI-negative; Q1-LR, Annexin-V-positive and PI-negative). (C) Statistical analysis of the ratio of apoptotic cells (Annexin-V-positive and PI-negative) or dead cells (PI-positive) in flow cytometry analysis data (mean ± SD; n = 3; NS P > 0.05, *** P < 0.001). P values were calculated by using two-way ANOVA. (D) The decline of cell viability induced by PRV infection cannot be recovered by Z-VAD-FMK treatment. PK-15 cells pretreated with 10 μM Z-VAD-FMK were mock-infected or infected with PRV GD-WH or RA strains (MOI = 10) for 24 h. Cell viability was analyzed by using the CCK8 assay as described in the Materials and Methods section (mean ± SD; n = 3; NS P > 0.05, ***P < 0.001). P values were calculated by using an unpaired Student’s t -test. (E) Western blot analysis showed that caspase proteins were not activated during PRV infection. PK-15 cells were mock-infected, infected with PRV GD-WH or RA strains (MOI = 10), or 50 nM staurosporine treated for 24 h. Caspase-3 and caspase-8 in whole cell lysates were analyzed by using Western blot as described in the Materials and Methods section.

    Techniques Used: Infection, Staining, Flow Cytometry, CCK-8 Assay, Western Blot

    Necrotic cell death during PRV infection was not related to RIPK1. (A) PRV infection raised the TNF-α transcription. PK-15 cells were mock-infected or infected with PRV GD-WH strain (MOI = 10) for 24 h. mRNA levels of TNF-α, FasL, and TraiL were analyzed by RT-qPCR as described in the Materials and Methods section (mean ± SD; n = 3; *P < 0.05, **P < 0.01). P values were calculated by using two-way ANOVA. (B) Western blot analysis protein expressions of TNF-α, FasL, and TraiL. PK-15 cells were mock-infected or infected with PRV GD-WH strain (MOI = 10) for 24 h. Protein expressions of TNF-α, FasL, and TraiL in whole cell lysates were analyzed by using Western blot as described in the Materials and Methods section. (C) Necrostatin-1 could not prevent the decline of cell viability during PRV infection. PK-15 cells pretreated with 100 μM necrostatin-1 were mock-infected or infected with PRV GD-WH strain (MOI = 10) for 24 h. Cell viability was analyzed by using the CCK8 assay as described in the Materials and Methods section (mean ± SD; n = 3; NS P > 0.05, ***P < 0.001). P values were calculated by using an unpaired Student’s t -test. (D) Necrostatin-1 had no effect on necrotic cells with PI staining induced by PRV infection. PK-15 cells pretreated with 100 μM necrostatin-1 were mock-infected or infected with PRV GD-WH strain (MOI = 10) for 24 h. PK-15 cells treated with TSZ (10 ng/ml TNF-α combined with 2 μM SM-164 and 10 μM Z-VAD-FMK) were used as a positive control of classical necroptosis. After cells were stained with PI (red), live images were snapped. (E) Statistical analysis of the ratio of PI-positive cells in live images (mean ± SD; n = 3; NS P > 0.05, ***P < 0.001). P values were calculated by using an unpaired Student’s t -test.
    Figure Legend Snippet: Necrotic cell death during PRV infection was not related to RIPK1. (A) PRV infection raised the TNF-α transcription. PK-15 cells were mock-infected or infected with PRV GD-WH strain (MOI = 10) for 24 h. mRNA levels of TNF-α, FasL, and TraiL were analyzed by RT-qPCR as described in the Materials and Methods section (mean ± SD; n = 3; *P < 0.05, **P < 0.01). P values were calculated by using two-way ANOVA. (B) Western blot analysis protein expressions of TNF-α, FasL, and TraiL. PK-15 cells were mock-infected or infected with PRV GD-WH strain (MOI = 10) for 24 h. Protein expressions of TNF-α, FasL, and TraiL in whole cell lysates were analyzed by using Western blot as described in the Materials and Methods section. (C) Necrostatin-1 could not prevent the decline of cell viability during PRV infection. PK-15 cells pretreated with 100 μM necrostatin-1 were mock-infected or infected with PRV GD-WH strain (MOI = 10) for 24 h. Cell viability was analyzed by using the CCK8 assay as described in the Materials and Methods section (mean ± SD; n = 3; NS P > 0.05, ***P < 0.001). P values were calculated by using an unpaired Student’s t -test. (D) Necrostatin-1 had no effect on necrotic cells with PI staining induced by PRV infection. PK-15 cells pretreated with 100 μM necrostatin-1 were mock-infected or infected with PRV GD-WH strain (MOI = 10) for 24 h. PK-15 cells treated with TSZ (10 ng/ml TNF-α combined with 2 μM SM-164 and 10 μM Z-VAD-FMK) were used as a positive control of classical necroptosis. After cells were stained with PI (red), live images were snapped. (E) Statistical analysis of the ratio of PI-positive cells in live images (mean ± SD; n = 3; NS P > 0.05, ***P < 0.001). P values were calculated by using an unpaired Student’s t -test.

    Techniques Used: Infection, Quantitative RT-PCR, Western Blot, CCK-8 Assay, Staining, Positive Control

    PRV enhanced the phosphorylation of RIPK3 and MLKL. (A) Western blot analysis showed that the phosphorylation of RIPK3 and MLKL was increased by PRV infection. PK-15 cells were mock-infected or infected with PRV GD-WH strain (MOI = 10) for 24 h. The phosphorylation or protein expression of RIPK3 and MLKL in whole cell lysates was analyzed by using Western blot as described in the Materials and Methods section. (B) Statistical analysis of the intensity of phosphorylation bands. Relative intensity of RIPKK3 or MLKL phosphorylation was obtained by comparing the densitometry of phosphorylation bands to its protein expression bands. GAPDH was used as an internal loading control (mean ± SD; n = 3; *P < 0.05, ***P < 0.001). P values were calculated by using two-way ANOVA. (C) PRV inhibited the transcription of RIPK3 and MLKL. PK-15 cells were mock-infected or infected with PRV GD-WH strain (MOI = 10) for 12 or 24 h. mRNA levels of RIPK3 and MLKL were analyzed by RT-qPCR as described in the Materials and Methods section (mean ± SD; n = 3; ***P < 0.001). P values were calculated by using two-way ANOVA.
    Figure Legend Snippet: PRV enhanced the phosphorylation of RIPK3 and MLKL. (A) Western blot analysis showed that the phosphorylation of RIPK3 and MLKL was increased by PRV infection. PK-15 cells were mock-infected or infected with PRV GD-WH strain (MOI = 10) for 24 h. The phosphorylation or protein expression of RIPK3 and MLKL in whole cell lysates was analyzed by using Western blot as described in the Materials and Methods section. (B) Statistical analysis of the intensity of phosphorylation bands. Relative intensity of RIPKK3 or MLKL phosphorylation was obtained by comparing the densitometry of phosphorylation bands to its protein expression bands. GAPDH was used as an internal loading control (mean ± SD; n = 3; *P < 0.05, ***P < 0.001). P values were calculated by using two-way ANOVA. (C) PRV inhibited the transcription of RIPK3 and MLKL. PK-15 cells were mock-infected or infected with PRV GD-WH strain (MOI = 10) for 12 or 24 h. mRNA levels of RIPK3 and MLKL were analyzed by RT-qPCR as described in the Materials and Methods section (mean ± SD; n = 3; ***P < 0.001). P values were calculated by using two-way ANOVA.

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

    Knockdown of RIPK3 and MLKL dampened PRV-induced necroptosis but increased viral titers. (A) qPCR analysis indicated that RIPK3 or MLKL transcription was down-regulated in PK-15 cells stably expressing shRNA. mRNA levels of RIPK3 and MLKL were analyzed by RT-qPCR as described in the Materials and Methods section (mean ± SD; n = 3; NS P > 0.05, *** P < 0.001). P values were calculated by using two-way ANOVA. (D) Knockdown of RIPK3 or MLKL prevented the reduction of cell viability during PRV infection. PK-15 cells stably expressing shRNA were mock-infected or infected with PRV GD-WH strain (MOI = 10) for 24 h. Cell viability was analyzed by using the CCK8 assay as described in the Materials and Methods section (mean ± SD; n = 3; NS P > 0.05, ***P < 0.001). P values were calculated by using an unpaired Student’s t -test. (B) RIPK3 or MLKL knockdown decreased PI-positive cells during PRV infection. PK-15 cells stably expressing shRNA were mock-infected or infected with PRV GD-WH strain (MOI = 10) for 24 h. After cells were stained with PI (red), live images were snapped. (C) Statistical analysis of the ratio of PI-positive cells in live images (mean ± SD; n = 3; NS P > 0.05, ***P < 0.001). P values were calculated by using an unpaired Student’s t -test. (E) Virus titers were increased in RIPK3 or MLKL knockdown PK-15 cells. PK-15 cells stably expressing shRNA were mock-infected or infected with PRV GD-WH strain (MOI = 1 or 10) for 12 or 24 h. Virus titers were analyzed as described in the Materials and Methods section (mean ± SD; n = 3; NS P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001). P values were calculated by using two-way ANOVA.
    Figure Legend Snippet: Knockdown of RIPK3 and MLKL dampened PRV-induced necroptosis but increased viral titers. (A) qPCR analysis indicated that RIPK3 or MLKL transcription was down-regulated in PK-15 cells stably expressing shRNA. mRNA levels of RIPK3 and MLKL were analyzed by RT-qPCR as described in the Materials and Methods section (mean ± SD; n = 3; NS P > 0.05, *** P < 0.001). P values were calculated by using two-way ANOVA. (D) Knockdown of RIPK3 or MLKL prevented the reduction of cell viability during PRV infection. PK-15 cells stably expressing shRNA were mock-infected or infected with PRV GD-WH strain (MOI = 10) for 24 h. Cell viability was analyzed by using the CCK8 assay as described in the Materials and Methods section (mean ± SD; n = 3; NS P > 0.05, ***P < 0.001). P values were calculated by using an unpaired Student’s t -test. (B) RIPK3 or MLKL knockdown decreased PI-positive cells during PRV infection. PK-15 cells stably expressing shRNA were mock-infected or infected with PRV GD-WH strain (MOI = 10) for 24 h. After cells were stained with PI (red), live images were snapped. (C) Statistical analysis of the ratio of PI-positive cells in live images (mean ± SD; n = 3; NS P > 0.05, ***P < 0.001). P values were calculated by using an unpaired Student’s t -test. (E) Virus titers were increased in RIPK3 or MLKL knockdown PK-15 cells. PK-15 cells stably expressing shRNA were mock-infected or infected with PRV GD-WH strain (MOI = 1 or 10) for 12 or 24 h. Virus titers were analyzed as described in the Materials and Methods section (mean ± SD; n = 3; NS P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001). P values were calculated by using two-way ANOVA.

    Techniques Used: Stable Transfection, Expressing, shRNA, Quantitative RT-PCR, Infection, CCK-8 Assay, Staining

    swine kidney cell line pk 15  (ATCC)


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    Structured Review

    ATCC swine kidney cell line pk 15
    (A) Growth kinetics of the variant virulent and classical attenuated pseudorabies virus (PRV) strains. <t>PK-15</t> cells were infected with the variant virulent (GD-WH) or classical attenuated (Bartha) PRV strains (MOI = 10), and the growth kinetics of each strain was analyzed as described in the Materials and Methods (mean ± SD; n=3; * P < 0.5; *** P < 0.001). P values were calculated using two-way ANOVA. (B) Cytopathic effects of PK-15 cells infected with the variant virulent or classical attenuated PRV strains at 24 hpi. PK-15 cells were infected with the variant virulent (GD-WH) or classical attenuated (Bartha) PRV strains (MOI=10). At 24 hpi, the cytopathic effects of PRV infection of PK-15 cells were observed (scale bars=400 μm).
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    1) Product Images from "Metabolomics Exploration of Pseudorabies Virus Reprogramming Metabolic Profiles of PK-15 Cells to Enhance Viral Replication"

    Article Title: Metabolomics Exploration of Pseudorabies Virus Reprogramming Metabolic Profiles of PK-15 Cells to Enhance Viral Replication

    Journal: Frontiers in Cellular and Infection Microbiology

    doi: 10.3389/fcimb.2020.599087

    (A) Growth kinetics of the variant virulent and classical attenuated pseudorabies virus (PRV) strains. PK-15 cells were infected with the variant virulent (GD-WH) or classical attenuated (Bartha) PRV strains (MOI = 10), and the growth kinetics of each strain was analyzed as described in the Materials and Methods (mean ± SD; n=3; * P < 0.5; *** P < 0.001). P values were calculated using two-way ANOVA. (B) Cytopathic effects of PK-15 cells infected with the variant virulent or classical attenuated PRV strains at 24 hpi. PK-15 cells were infected with the variant virulent (GD-WH) or classical attenuated (Bartha) PRV strains (MOI=10). At 24 hpi, the cytopathic effects of PRV infection of PK-15 cells were observed (scale bars=400 μm).
    Figure Legend Snippet: (A) Growth kinetics of the variant virulent and classical attenuated pseudorabies virus (PRV) strains. PK-15 cells were infected with the variant virulent (GD-WH) or classical attenuated (Bartha) PRV strains (MOI = 10), and the growth kinetics of each strain was analyzed as described in the Materials and Methods (mean ± SD; n=3; * P < 0.5; *** P < 0.001). P values were calculated using two-way ANOVA. (B) Cytopathic effects of PK-15 cells infected with the variant virulent or classical attenuated PRV strains at 24 hpi. PK-15 cells were infected with the variant virulent (GD-WH) or classical attenuated (Bartha) PRV strains (MOI=10). At 24 hpi, the cytopathic effects of PRV infection of PK-15 cells were observed (scale bars=400 μm).

    Techniques Used: Variant Assay, Infection

    Heatmap visualization of differential metabolites in PK-15 cells infected with the variant virulent (A, B) or classical attenuated (C, D) PRV strains at 8 and 16 hpi. Rows: metabolites; columns: samples. The color from red (positive value) to green (negative value) of each rectangle was based on the ratio between pseudorabies virus (PRV)-infected groups vs mock groups. For example, a red color means that the average mass response of the metabolite in PRV-infected groups was greater than that in mock groups. In all images, W8 and W16 represent the groups infected with the variant virulent (GD-WH) strain; B8 and B16 represent the groups infected with the classical attenuated (Bartha) strain, and M8 and M16 represent the mock groups.
    Figure Legend Snippet: Heatmap visualization of differential metabolites in PK-15 cells infected with the variant virulent (A, B) or classical attenuated (C, D) PRV strains at 8 and 16 hpi. Rows: metabolites; columns: samples. The color from red (positive value) to green (negative value) of each rectangle was based on the ratio between pseudorabies virus (PRV)-infected groups vs mock groups. For example, a red color means that the average mass response of the metabolite in PRV-infected groups was greater than that in mock groups. In all images, W8 and W16 represent the groups infected with the variant virulent (GD-WH) strain; B8 and B16 represent the groups infected with the classical attenuated (Bartha) strain, and M8 and M16 represent the mock groups.

    Techniques Used: Infection, Variant Assay

    Statistical analysis of fold change of differential metabolites in PK-15 cells infected with the variant virulent (A, C, E, G) or classical attenuated (B, D, F, H) pseudorabies virus (PRV) strains at 8 and 16 hpi (mean ± SD; n=4). Fold change was calculated as a binary logarithm of the average mass response (normalized peak area) ratio between PRV-infected groups vs mock groups, where a positive value means that the average mass response of the metabolite in PRV-infected groups was greater than that in mock groups.
    Figure Legend Snippet: Statistical analysis of fold change of differential metabolites in PK-15 cells infected with the variant virulent (A, C, E, G) or classical attenuated (B, D, F, H) pseudorabies virus (PRV) strains at 8 and 16 hpi (mean ± SD; n=4). Fold change was calculated as a binary logarithm of the average mass response (normalized peak area) ratio between PRV-infected groups vs mock groups, where a positive value means that the average mass response of the metabolite in PRV-infected groups was greater than that in mock groups.

    Techniques Used: Infection, Variant Assay

    Inhibition of the glycolysis reduced the replication of pseudorabies virus (PRV) in PK-15 cells. (A) The effect of 2-deoxyglucose (2DG) treatment on the virus titers in PK-15 cells infected with the variant virulent PRV strain. PK-15 cells were pretreated with 10 or 20 mM 2DG for 3 h. Then, cells were infected with PRV GD-WH strain at an multiplicity of infection (MOI) of 1 and cultured in Dulbecco’s modified Eagle medium (DMEM) containing 10 or 20 mM 2DG. At 16 hpi, the titers of virus were analyzed as described in Materials and Methods (mean ± SD; n=3; ** P <0.01; *** P < 0.001). P values were calculated by using an unpaired Student’s t-test. (B) The effect of 2DG treatment on the cell viability of PK-15 cells. The cell viability of PK-15 cells treated with 10 and 20 mM 2DG was analyzed by the CCK8 assay as described in Materials and Methods (mean ± SD; n=3; NS P >0.05). (C) Glucose depletion or oxamate treatment decreased PRV titers in PK-15 cells. PK-15 cells were starved by be cultured in depletion DMEM repleted with 2 mM L-glutamine or pretreated with 50 mM oxamate for 3 h. Then cells were infected with PRV GD-WH strain at a MOI of 1. After being cultured in depletion DMEM repleted with 2 mM L-glutamine or DMEM containing 50 mM oxamate for 16 h, virus titers were analyzed as described in Materials and Methods (mean ± SD; n = 3; * P < 0.05; ** P < 0.01; *** P < 0.001). P values were calculated by using an unpaired Student’s t-test. (D) The effect of oxamate treatment on the cell viability of PK-15 cells. The cell viability of PK-15cells starved by glucose depletion or treated with oxamate for 16 h were analyzed by the CCK8 assay as described in Materials and Methods (mean ± SD; n = 3; NS P > 0.05).
    Figure Legend Snippet: Inhibition of the glycolysis reduced the replication of pseudorabies virus (PRV) in PK-15 cells. (A) The effect of 2-deoxyglucose (2DG) treatment on the virus titers in PK-15 cells infected with the variant virulent PRV strain. PK-15 cells were pretreated with 10 or 20 mM 2DG for 3 h. Then, cells were infected with PRV GD-WH strain at an multiplicity of infection (MOI) of 1 and cultured in Dulbecco’s modified Eagle medium (DMEM) containing 10 or 20 mM 2DG. At 16 hpi, the titers of virus were analyzed as described in Materials and Methods (mean ± SD; n=3; ** P <0.01; *** P < 0.001). P values were calculated by using an unpaired Student’s t-test. (B) The effect of 2DG treatment on the cell viability of PK-15 cells. The cell viability of PK-15 cells treated with 10 and 20 mM 2DG was analyzed by the CCK8 assay as described in Materials and Methods (mean ± SD; n=3; NS P >0.05). (C) Glucose depletion or oxamate treatment decreased PRV titers in PK-15 cells. PK-15 cells were starved by be cultured in depletion DMEM repleted with 2 mM L-glutamine or pretreated with 50 mM oxamate for 3 h. Then cells were infected with PRV GD-WH strain at a MOI of 1. After being cultured in depletion DMEM repleted with 2 mM L-glutamine or DMEM containing 50 mM oxamate for 16 h, virus titers were analyzed as described in Materials and Methods (mean ± SD; n = 3; * P < 0.05; ** P < 0.01; *** P < 0.001). P values were calculated by using an unpaired Student’s t-test. (D) The effect of oxamate treatment on the cell viability of PK-15 cells. The cell viability of PK-15cells starved by glucose depletion or treated with oxamate for 16 h were analyzed by the CCK8 assay as described in Materials and Methods (mean ± SD; n = 3; NS P > 0.05).

    Techniques Used: Inhibition, Infection, Variant Assay, Cell Culture, Modification, CCK-8 Assay

    The effect of the TCA cycle and pentose phosphate pathway (PPP) on pseudorabies virus (PRV) replication in PK-15 cells. (A) The effect of oligomycin A or 6-AN treatment on PRV replication. PK-15 cells were pretreated with 1 μM Oligomycin A or 500 μM 6-AN for 3 h. Then cells were infected with PRV GD-WH strain at a MOI of 1 and cultured in DMEM containing 1 μM Oligomycin A or 500 μM 6-AN. At 16 hpi, the titers of virus were analyzed as described in Materials and Methods (mean ± SD; n = 3; * P < 0.05; *** P < 0.001). P values were calculated by using an unpaired Student’s t-test. (B) The effect of Oligomycin A or 6-AN treatment on the cell viability of PK-15 cells. The cell viability of PK-15 cells treated with 1 μM Oligomycin A or 500 μM 6-AN were analyzed by the CCK8 assay as described in Materials and Methods (mean ± SD; n = 3; NS P > 0.05).
    Figure Legend Snippet: The effect of the TCA cycle and pentose phosphate pathway (PPP) on pseudorabies virus (PRV) replication in PK-15 cells. (A) The effect of oligomycin A or 6-AN treatment on PRV replication. PK-15 cells were pretreated with 1 μM Oligomycin A or 500 μM 6-AN for 3 h. Then cells were infected with PRV GD-WH strain at a MOI of 1 and cultured in DMEM containing 1 μM Oligomycin A or 500 μM 6-AN. At 16 hpi, the titers of virus were analyzed as described in Materials and Methods (mean ± SD; n = 3; * P < 0.05; *** P < 0.001). P values were calculated by using an unpaired Student’s t-test. (B) The effect of Oligomycin A or 6-AN treatment on the cell viability of PK-15 cells. The cell viability of PK-15 cells treated with 1 μM Oligomycin A or 500 μM 6-AN were analyzed by the CCK8 assay as described in Materials and Methods (mean ± SD; n = 3; NS P > 0.05).

    Techniques Used: Infection, Cell Culture, CCK-8 Assay

    Depletion of glutamine reduced pseudorabies virus (PRV) replication in PK-15 cells in a manner independent of the TCA cycle. (A) Glutamine starvation had a repressive effect on PRV replication in PK-15 cells. PK-15 cells were starved by being cultured in glutamine-free DMEM for 3 h. Then, cells were infected with PRV GD-WH strain at an multiplicity of infection (MOI) of 1 and cultured in normal Dulbecco’s modified Eagle medium (DMEM) or no glutamine DMEM. In replenishment groups, 2 or 4 mM L-glutamine was added to glutamine-free DMEM. At 16 h, the titers of virus were analyzed as described in Materials and Methods (mean ± SD; n=3; * P <0.05; ** P <0.01; *** P < 0.001). P values were calculated by using an unpaired Student’s t-test. (B) BPTES treatment had no effect on PRV replication in PK-15 cells. PK-15 cells were pretreated with 1 μM BPTES for 3 h. Then cells were infected with PRV GD-WH strain at a MOI of 1 and cultured in DMEM containing 1 μM BPTES. At 16 hpi, the titers of virus were analyzed as described in Materials and Methods (mean ± SD; n = 3; NS P > 0.05; ** P < 0.01). P values were calculated by using an unpaired Student’s t-test. (C) The effect of BPTES treatment on the cell viability of PK-15 cells. The cell viability of PK-15cells treated with 1 μM BPTES were analyzed by the CCK8 assay as described in Materials and Methods (mean ± SD; n = 3; NS P > 0.05). (C) 2-ketoglutarate supplement cannot recover the effect of glutamine starvation on PRV replication in PK-15 cells. PK-15 cells were starved by being cultured in no glutamine DMEM for 3 h. Then cells were infected with PRV GD-WH strain at a MOI of 1 and cultured in normal DMEM or no glutamine DMEM. In 2-ketoglutarate supplement groups, 5 mM 2-ketoglutarate was added in no glutamine DMEM. At 16 h, the titers of virus were analyzed as described in Materials and Methods (mean ± SD; n = 3; NS P > 0.05). P values were calculated by using an unpaired Student’s t-test.
    Figure Legend Snippet: Depletion of glutamine reduced pseudorabies virus (PRV) replication in PK-15 cells in a manner independent of the TCA cycle. (A) Glutamine starvation had a repressive effect on PRV replication in PK-15 cells. PK-15 cells were starved by being cultured in glutamine-free DMEM for 3 h. Then, cells were infected with PRV GD-WH strain at an multiplicity of infection (MOI) of 1 and cultured in normal Dulbecco’s modified Eagle medium (DMEM) or no glutamine DMEM. In replenishment groups, 2 or 4 mM L-glutamine was added to glutamine-free DMEM. At 16 h, the titers of virus were analyzed as described in Materials and Methods (mean ± SD; n=3; * P <0.05; ** P <0.01; *** P < 0.001). P values were calculated by using an unpaired Student’s t-test. (B) BPTES treatment had no effect on PRV replication in PK-15 cells. PK-15 cells were pretreated with 1 μM BPTES for 3 h. Then cells were infected with PRV GD-WH strain at a MOI of 1 and cultured in DMEM containing 1 μM BPTES. At 16 hpi, the titers of virus were analyzed as described in Materials and Methods (mean ± SD; n = 3; NS P > 0.05; ** P < 0.01). P values were calculated by using an unpaired Student’s t-test. (C) The effect of BPTES treatment on the cell viability of PK-15 cells. The cell viability of PK-15cells treated with 1 μM BPTES were analyzed by the CCK8 assay as described in Materials and Methods (mean ± SD; n = 3; NS P > 0.05). (C) 2-ketoglutarate supplement cannot recover the effect of glutamine starvation on PRV replication in PK-15 cells. PK-15 cells were starved by being cultured in no glutamine DMEM for 3 h. Then cells were infected with PRV GD-WH strain at a MOI of 1 and cultured in normal DMEM or no glutamine DMEM. In 2-ketoglutarate supplement groups, 5 mM 2-ketoglutarate was added in no glutamine DMEM. At 16 h, the titers of virus were analyzed as described in Materials and Methods (mean ± SD; n = 3; NS P > 0.05). P values were calculated by using an unpaired Student’s t-test.

    Techniques Used: Cell Culture, Infection, Modification, CCK-8 Assay

    Schematic overview of metabolic pathways in PK-15 cells infected by different pseudorabies virus (PRV) strains at 8 (A) and 16 (B) hpi. The metabolites are shown in different colors according to their changes: red indicates increased metabolites; green indicates decreased metabolites, and white indicates no difference metabolites.
    Figure Legend Snippet: Schematic overview of metabolic pathways in PK-15 cells infected by different pseudorabies virus (PRV) strains at 8 (A) and 16 (B) hpi. The metabolites are shown in different colors according to their changes: red indicates increased metabolites; green indicates decreased metabolites, and white indicates no difference metabolites.

    Techniques Used: Infection

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    ATCC swine kidney cell line pk 15
    FMDV infection-induced autophagy plays an important role in viral replication. ( A ) <t>PK-15</t> cells were mock infected or infected with FMDV (MOI = 1) for 0, 0.5, 1, 1.5, 2 and 3 h. The expression of LC3B and SQSTM1 was analyzed by western blot. The level of 3D was analyzed by RT-PCR. ACTB and GAPDH were used as a sample loading control. ( B ) The cells were then fixed and processed for indirect immunofluorescence using antibodies against LC3B and the 3D protein, followed by the corresponding secondary antibodies. The fluorescence signals were visualized by confocal immunofluorescence microscopy. ( C ) ATG5 knockdown (KD) and wild-type cells were infected with FMDV (MOI = 1) for 2 and 3 h. The expression of ATG5 and LC3B was analyzed by western blot. ( D and E ) ATG5 KD and wild-type cells were infected with FMDV (MOI = 1). At 3 hpi, both the extracellular and intracellular copy numbers of FMDV were measured by qRT-PCR; both the extracellular and intracellular virus titers were measured by TCID 50 . The data represent the mean ± SD of 3 independent experiments. ***P < 0.001. ( F ) PK-15 cells were mock infected or infected with FMDV (MOI = 1) for 6 and 9 h. The expression of SQSTM1 and VP1 were analyzed by western blot. ( G ) Cells were transfected with pmCherry-GFP-LC3B for 24 h, followed by FMDV infection (MOI = 1) and treatment with Baf-A1. The fluorescence signals were visualized by confocal immunofluorescence microscopy. Scale bars: 10 μm.
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    FMDV infection-induced autophagy plays an important role in viral replication. ( A ) PK-15 cells were mock infected or infected with FMDV (MOI = 1) for 0, 0.5, 1, 1.5, 2 and 3 h. The expression of LC3B and SQSTM1 was analyzed by western blot. The level of 3D was analyzed by RT-PCR. ACTB and GAPDH were used as a sample loading control. ( B ) The cells were then fixed and processed for indirect immunofluorescence using antibodies against LC3B and the 3D protein, followed by the corresponding secondary antibodies. The fluorescence signals were visualized by confocal immunofluorescence microscopy. ( C ) ATG5 knockdown (KD) and wild-type cells were infected with FMDV (MOI = 1) for 2 and 3 h. The expression of ATG5 and LC3B was analyzed by western blot. ( D and E ) ATG5 KD and wild-type cells were infected with FMDV (MOI = 1). At 3 hpi, both the extracellular and intracellular copy numbers of FMDV were measured by qRT-PCR; both the extracellular and intracellular virus titers were measured by TCID 50 . The data represent the mean ± SD of 3 independent experiments. ***P < 0.001. ( F ) PK-15 cells were mock infected or infected with FMDV (MOI = 1) for 6 and 9 h. The expression of SQSTM1 and VP1 were analyzed by western blot. ( G ) Cells were transfected with pmCherry-GFP-LC3B for 24 h, followed by FMDV infection (MOI = 1) and treatment with Baf-A1. The fluorescence signals were visualized by confocal immunofluorescence microscopy. Scale bars: 10 μm.

    Journal: Autophagy

    Article Title: Foot-and-mouth disease virus capsid protein VP2 activates the cellular EIF2S1-ATF4 pathway and induces autophagy via HSPB1

    doi: 10.1080/15548627.2017.1405187

    Figure Lengend Snippet: FMDV infection-induced autophagy plays an important role in viral replication. ( A ) PK-15 cells were mock infected or infected with FMDV (MOI = 1) for 0, 0.5, 1, 1.5, 2 and 3 h. The expression of LC3B and SQSTM1 was analyzed by western blot. The level of 3D was analyzed by RT-PCR. ACTB and GAPDH were used as a sample loading control. ( B ) The cells were then fixed and processed for indirect immunofluorescence using antibodies against LC3B and the 3D protein, followed by the corresponding secondary antibodies. The fluorescence signals were visualized by confocal immunofluorescence microscopy. ( C ) ATG5 knockdown (KD) and wild-type cells were infected with FMDV (MOI = 1) for 2 and 3 h. The expression of ATG5 and LC3B was analyzed by western blot. ( D and E ) ATG5 KD and wild-type cells were infected with FMDV (MOI = 1). At 3 hpi, both the extracellular and intracellular copy numbers of FMDV were measured by qRT-PCR; both the extracellular and intracellular virus titers were measured by TCID 50 . The data represent the mean ± SD of 3 independent experiments. ***P < 0.001. ( F ) PK-15 cells were mock infected or infected with FMDV (MOI = 1) for 6 and 9 h. The expression of SQSTM1 and VP1 were analyzed by western blot. ( G ) Cells were transfected with pmCherry-GFP-LC3B for 24 h, followed by FMDV infection (MOI = 1) and treatment with Baf-A1. The fluorescence signals were visualized by confocal immunofluorescence microscopy. Scale bars: 10 μm.

    Article Snippet: The swine kidney cell line PK-15 (ATCC, CCL-33), HEK293T (ATCC, CRL-11268) and Vero (ATCC, CCL-81) cells were maintained in complete Dulbecco's modified Eagle's medium (Gibco, C11995500BT) supplemented with 10% fetal bovine serum and 1% antibiotics.

    Techniques: Infection, Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Fluorescence, Microscopy, Quantitative RT-PCR, Transfection

    FMDV infection induces autophagy through the EIF2S1-ATF4-AKT-MTOR cascade. ( A ) PK-15 cells were mock infected or infected with FMDV (MOI = 1) for 1.5, 2 and 3 h. The phosphorylation of AKT, AMPK, MTOR and ULK1 were analyzed by western blot. ACTB was used as a sample loading control. ( B ) PK-15 cells were mock infected or infected with FMDV (MOI = 1) for 1.5, 2 and 3 h. ATF4 and phosphorylation of EIF2S1 were analyzed by western blot. ( C ) ATF4 KD and wild-type cells cells were infected with FMDV (MOI = 1). ATF4, LC3B and phosphorylation of AKT, MTOR and ULK1 were analyzed by western blot. ( D ) ATF4 and scrambled knockdown cells were transfected with pmCherry-GFP-LC3B for 24 h, followed by FMDV infection (MOI = 1) for 3 h. The fluorescence signals were visualized by confocal immunofluorescence microscopy. Scale bars: 10 μm. ( E and F ) ATF4 KD and wil-type cells cells were infected with FMDV (MOI = 1) for 3 h. At 3 hpi, both the extracellular and intracellular copy numbers of FMDV were measured by qRT-PCR; both the extracellular and intracellular virus titers were measured by TCID 50 . The data represent the mean ± SD of 3 independent experiments. **P < 0.01, ***P < 0.001.

    Journal: Autophagy

    Article Title: Foot-and-mouth disease virus capsid protein VP2 activates the cellular EIF2S1-ATF4 pathway and induces autophagy via HSPB1

    doi: 10.1080/15548627.2017.1405187

    Figure Lengend Snippet: FMDV infection induces autophagy through the EIF2S1-ATF4-AKT-MTOR cascade. ( A ) PK-15 cells were mock infected or infected with FMDV (MOI = 1) for 1.5, 2 and 3 h. The phosphorylation of AKT, AMPK, MTOR and ULK1 were analyzed by western blot. ACTB was used as a sample loading control. ( B ) PK-15 cells were mock infected or infected with FMDV (MOI = 1) for 1.5, 2 and 3 h. ATF4 and phosphorylation of EIF2S1 were analyzed by western blot. ( C ) ATF4 KD and wild-type cells cells were infected with FMDV (MOI = 1). ATF4, LC3B and phosphorylation of AKT, MTOR and ULK1 were analyzed by western blot. ( D ) ATF4 and scrambled knockdown cells were transfected with pmCherry-GFP-LC3B for 24 h, followed by FMDV infection (MOI = 1) for 3 h. The fluorescence signals were visualized by confocal immunofluorescence microscopy. Scale bars: 10 μm. ( E and F ) ATF4 KD and wil-type cells cells were infected with FMDV (MOI = 1) for 3 h. At 3 hpi, both the extracellular and intracellular copy numbers of FMDV were measured by qRT-PCR; both the extracellular and intracellular virus titers were measured by TCID 50 . The data represent the mean ± SD of 3 independent experiments. **P < 0.01, ***P < 0.001.

    Article Snippet: The swine kidney cell line PK-15 (ATCC, CCL-33), HEK293T (ATCC, CRL-11268) and Vero (ATCC, CCL-81) cells were maintained in complete Dulbecco's modified Eagle's medium (Gibco, C11995500BT) supplemented with 10% fetal bovine serum and 1% antibiotics.

    Techniques: Infection, Western Blot, Transfection, Fluorescence, Immunofluorescence, Microscopy, Quantitative RT-PCR

    UV-FMDV infection induces autophagy. ( A ) PK-15 cells were mock infected or infected with UV-FMDV for 3 h (MOI = 10). LC3B and phosphorylation of EIF2S1, AKT and MTOR were analyzed by western blot. ACTB was used as a sample loading control. ( B ) ATF4 KD and wild-type cells were infected as described in ( A ).

    Journal: Autophagy

    Article Title: Foot-and-mouth disease virus capsid protein VP2 activates the cellular EIF2S1-ATF4 pathway and induces autophagy via HSPB1

    doi: 10.1080/15548627.2017.1405187

    Figure Lengend Snippet: UV-FMDV infection induces autophagy. ( A ) PK-15 cells were mock infected or infected with UV-FMDV for 3 h (MOI = 10). LC3B and phosphorylation of EIF2S1, AKT and MTOR were analyzed by western blot. ACTB was used as a sample loading control. ( B ) ATF4 KD and wild-type cells were infected as described in ( A ).

    Article Snippet: The swine kidney cell line PK-15 (ATCC, CCL-33), HEK293T (ATCC, CRL-11268) and Vero (ATCC, CCL-81) cells were maintained in complete Dulbecco's modified Eagle's medium (Gibco, C11995500BT) supplemented with 10% fetal bovine serum and 1% antibiotics.

    Techniques: Infection, Western Blot

    FMDV capsid protein VP2 induced autophagy. ( A ) PK-15 cells were transfected with empty vectors or various plasmids expressing FLAG-tagged VP1, VP2and VP3 proteins for 24 h. LC3B and FMDV capsid proteins were analyzed by western blot. ACTB was used as a sample loading control. ( B ) Cells were transfected with empty vectors or pCMV-Flag-VP2 for 24 h. Cells were fixed and analyzed by immunofluorescence using anti-LC3B antibodies. The fluorescence signals were visualized by confocal immunofluorescence microscopy. Scale bars: 10 μm. ( C ) PK-15 cells were transfected with empty vectors or pCMV-Flag-VP2 for 24 h. LC3B and phosphorylation of EIF2S1, AKT and MTOR were analyzed by western blot. ACTB was used as a sample loading control. ( D ) ATF4 and scrambled knockdown cells were transfected with pCMV-Flag-VP2 for 24 h. LC3B and phosphorylation of AKT and MTOR were analyzed by western blot. ACTB was used as a sample loading control.

    Journal: Autophagy

    Article Title: Foot-and-mouth disease virus capsid protein VP2 activates the cellular EIF2S1-ATF4 pathway and induces autophagy via HSPB1

    doi: 10.1080/15548627.2017.1405187

    Figure Lengend Snippet: FMDV capsid protein VP2 induced autophagy. ( A ) PK-15 cells were transfected with empty vectors or various plasmids expressing FLAG-tagged VP1, VP2and VP3 proteins for 24 h. LC3B and FMDV capsid proteins were analyzed by western blot. ACTB was used as a sample loading control. ( B ) Cells were transfected with empty vectors or pCMV-Flag-VP2 for 24 h. Cells were fixed and analyzed by immunofluorescence using anti-LC3B antibodies. The fluorescence signals were visualized by confocal immunofluorescence microscopy. Scale bars: 10 μm. ( C ) PK-15 cells were transfected with empty vectors or pCMV-Flag-VP2 for 24 h. LC3B and phosphorylation of EIF2S1, AKT and MTOR were analyzed by western blot. ACTB was used as a sample loading control. ( D ) ATF4 and scrambled knockdown cells were transfected with pCMV-Flag-VP2 for 24 h. LC3B and phosphorylation of AKT and MTOR were analyzed by western blot. ACTB was used as a sample loading control.

    Article Snippet: The swine kidney cell line PK-15 (ATCC, CCL-33), HEK293T (ATCC, CRL-11268) and Vero (ATCC, CCL-81) cells were maintained in complete Dulbecco's modified Eagle's medium (Gibco, C11995500BT) supplemented with 10% fetal bovine serum and 1% antibiotics.

    Techniques: Transfection, Expressing, Western Blot, Immunofluorescence, Fluorescence, Microscopy

    VP2 mutant and interaction between VP2 and HSPB1 in PK-15 cells. ( A ) A scheme of the VP2 muant. ( B ) PK-15 cells were transfected with empty vectors, pCMV-Flag-VP2, or VP2 mutant for 24 h. LC3B and ACTB were analyzed by western blot. ( C and D ) The interaction between FMDV VP2 and HSPB1 in PK-15 cells was verified. The PK-15 cells were co-transfected with 10 μg pCMV-Flag-VP2 plasmid and 10 μg pEGFP-HA-HSPB1 plasmid or transfected with just 10 μg pCMV-Flag-VP2 plasmid, and immunoprecipitation was performed with anti-HA antibody. Immunoblotting analysis was performed with anti-HA antibody and anti-FLAG antibody. ( E and F ) The PK-15 cells were transfected with 10 μg pCMV-Flag-VP2 plasmid or 10 μg empty vector pCMV-Flag plasmid, and immunoprecipitation was performed with anti-FLAG antibody. Immunoblotting analysis was performed with anti-FLAG antibody and anti-HSPB1 antibody.

    Journal: Autophagy

    Article Title: Foot-and-mouth disease virus capsid protein VP2 activates the cellular EIF2S1-ATF4 pathway and induces autophagy via HSPB1

    doi: 10.1080/15548627.2017.1405187

    Figure Lengend Snippet: VP2 mutant and interaction between VP2 and HSPB1 in PK-15 cells. ( A ) A scheme of the VP2 muant. ( B ) PK-15 cells were transfected with empty vectors, pCMV-Flag-VP2, or VP2 mutant for 24 h. LC3B and ACTB were analyzed by western blot. ( C and D ) The interaction between FMDV VP2 and HSPB1 in PK-15 cells was verified. The PK-15 cells were co-transfected with 10 μg pCMV-Flag-VP2 plasmid and 10 μg pEGFP-HA-HSPB1 plasmid or transfected with just 10 μg pCMV-Flag-VP2 plasmid, and immunoprecipitation was performed with anti-HA antibody. Immunoblotting analysis was performed with anti-HA antibody and anti-FLAG antibody. ( E and F ) The PK-15 cells were transfected with 10 μg pCMV-Flag-VP2 plasmid or 10 μg empty vector pCMV-Flag plasmid, and immunoprecipitation was performed with anti-FLAG antibody. Immunoblotting analysis was performed with anti-FLAG antibody and anti-HSPB1 antibody.

    Article Snippet: The swine kidney cell line PK-15 (ATCC, CCL-33), HEK293T (ATCC, CRL-11268) and Vero (ATCC, CCL-81) cells were maintained in complete Dulbecco's modified Eagle's medium (Gibco, C11995500BT) supplemented with 10% fetal bovine serum and 1% antibiotics.

    Techniques: Mutagenesis, Transfection, Western Blot, Plasmid Preparation, Immunoprecipitation

    VP2 decreases aggregation of HTT103Q. ( A ) HEK293T cells and ( B) Vero cells were transfected with empty vectors or pCMV-N-VP2 for 24 h. LC3B and SQSTM1 by were analyzed by western blot. ACTB was used as a sample loading control. ( C ) PK-15 cells were transfected with empty vectors or pCMV-EGFP-HTT103Q for 24 h. HTT103Q and SQSTM1 were analyzed by western blot. ACTB was used as a sample loading control. ( D ) PK-15 cells and ATG5 KD cells were co-transfected with pCMV-Flag-VP2 and pCMV-EGFP-HTT103Q for 24 h. The fluorescence signals were visualized by confocal immunofluorescence microscopy. Scale bars: 10 μm. ( E ) The number of HTT103Q dot was counted. The data represent the mean ± SD of 3 independent experiments. **P < 0.01.

    Journal: Autophagy

    Article Title: Foot-and-mouth disease virus capsid protein VP2 activates the cellular EIF2S1-ATF4 pathway and induces autophagy via HSPB1

    doi: 10.1080/15548627.2017.1405187

    Figure Lengend Snippet: VP2 decreases aggregation of HTT103Q. ( A ) HEK293T cells and ( B) Vero cells were transfected with empty vectors or pCMV-N-VP2 for 24 h. LC3B and SQSTM1 by were analyzed by western blot. ACTB was used as a sample loading control. ( C ) PK-15 cells were transfected with empty vectors or pCMV-EGFP-HTT103Q for 24 h. HTT103Q and SQSTM1 were analyzed by western blot. ACTB was used as a sample loading control. ( D ) PK-15 cells and ATG5 KD cells were co-transfected with pCMV-Flag-VP2 and pCMV-EGFP-HTT103Q for 24 h. The fluorescence signals were visualized by confocal immunofluorescence microscopy. Scale bars: 10 μm. ( E ) The number of HTT103Q dot was counted. The data represent the mean ± SD of 3 independent experiments. **P < 0.01.

    Article Snippet: The swine kidney cell line PK-15 (ATCC, CCL-33), HEK293T (ATCC, CRL-11268) and Vero (ATCC, CCL-81) cells were maintained in complete Dulbecco's modified Eagle's medium (Gibco, C11995500BT) supplemented with 10% fetal bovine serum and 1% antibiotics.

    Techniques: Transfection, Western Blot, Fluorescence, Immunofluorescence, Microscopy