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 lines sk6  (ATCC)


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    ATCC swine kidney cell lines sk6
    Swine Kidney Cell Lines Sk6, 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 lines sk6  (ATCC)


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    ATCC swine kidney cell lines sk6
    Treatment with the cGAS inhibitor G140 promotes FMDV infection. a IBRS-2 cells (2 × 10 5 ) were infected with FMDV O1K at an MOI of 0.01. One hour later, infection medium was replaced with fresh medium containing 20 µM G140 and cells were further incubated for 18 h. Then, supernatants were collected and cells were fixed for VP1 expression analysis (red) in comparison with non-treated cells under a wide field fluorescence microscope (left panel); scale bars, show 100 µm. Viral titers in the supernatants (tenfold dilutions) were quantified by plaque assay (right panel). b , c , d IBRS-2 ( b ), <t>SK6</t> ( c ) or WSL ( d ) cells were infected and treated with increasing concentrations of G140 as in ( a ) and 18 h later the supernatants were collected and viral titers were determined by plaque assay. Data are mean ± SD of at least three independent assays performed in duplicate. e HEK293T cells (0.4 × 10 6 ) were co-transfected with 25 ng of pIFN-β-FL, 12.5 ng of pRL-TK and either 2 ng of 2CARD or po cGAS/ h STING (10 ng each). After 7 h, G140 was added to the medium at the indicated concentrations and cells were incubated for 24 h. Then, cells were harvested for IFN-β assay. Data are mean ± SD of three independent assays. Significant differences (one-way ANOVA, p values) are indicated
    Swine Kidney Cell Lines Sk6, 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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    1) Product Images from "The antiviral response triggered by the cGAS/STING pathway is subverted by the foot-and-mouth disease virus proteases"

    Article Title: The antiviral response triggered by the cGAS/STING pathway is subverted by the foot-and-mouth disease virus proteases

    Journal: Cellular and Molecular Life Sciences

    doi: 10.1007/s00018-024-05190-7

    Treatment with the cGAS inhibitor G140 promotes FMDV infection. a IBRS-2 cells (2 × 10 5 ) were infected with FMDV O1K at an MOI of 0.01. One hour later, infection medium was replaced with fresh medium containing 20 µM G140 and cells were further incubated for 18 h. Then, supernatants were collected and cells were fixed for VP1 expression analysis (red) in comparison with non-treated cells under a wide field fluorescence microscope (left panel); scale bars, show 100 µm. Viral titers in the supernatants (tenfold dilutions) were quantified by plaque assay (right panel). b , c , d IBRS-2 ( b ), SK6 ( c ) or WSL ( d ) cells were infected and treated with increasing concentrations of G140 as in ( a ) and 18 h later the supernatants were collected and viral titers were determined by plaque assay. Data are mean ± SD of at least three independent assays performed in duplicate. e HEK293T cells (0.4 × 10 6 ) were co-transfected with 25 ng of pIFN-β-FL, 12.5 ng of pRL-TK and either 2 ng of 2CARD or po cGAS/ h STING (10 ng each). After 7 h, G140 was added to the medium at the indicated concentrations and cells were incubated for 24 h. Then, cells were harvested for IFN-β assay. Data are mean ± SD of three independent assays. Significant differences (one-way ANOVA, p values) are indicated
    Figure Legend Snippet: Treatment with the cGAS inhibitor G140 promotes FMDV infection. a IBRS-2 cells (2 × 10 5 ) were infected with FMDV O1K at an MOI of 0.01. One hour later, infection medium was replaced with fresh medium containing 20 µM G140 and cells were further incubated for 18 h. Then, supernatants were collected and cells were fixed for VP1 expression analysis (red) in comparison with non-treated cells under a wide field fluorescence microscope (left panel); scale bars, show 100 µm. Viral titers in the supernatants (tenfold dilutions) were quantified by plaque assay (right panel). b , c , d IBRS-2 ( b ), SK6 ( c ) or WSL ( d ) cells were infected and treated with increasing concentrations of G140 as in ( a ) and 18 h later the supernatants were collected and viral titers were determined by plaque assay. Data are mean ± SD of at least three independent assays performed in duplicate. e HEK293T cells (0.4 × 10 6 ) were co-transfected with 25 ng of pIFN-β-FL, 12.5 ng of pRL-TK and either 2 ng of 2CARD or po cGAS/ h STING (10 ng each). After 7 h, G140 was added to the medium at the indicated concentrations and cells were incubated for 24 h. Then, cells were harvested for IFN-β assay. Data are mean ± SD of three independent assays. Significant differences (one-way ANOVA, p values) are indicated

    Techniques Used: Infection, Incubation, Expressing, Comparison, Fluorescence, Microscopy, Plaque Assay, Transfection

    FMDV infection is enhanced by treatment with the gap-junction inhibitor carbenoxolone (CBX). IBRS-2 ( a ), SK6 ( b ) or WSL ( c ) cells (2 × 10 5 ) were treated with CBX at different concentrations for 3 h and then infected with FMDV O1K at an MOI of 0.01. Supernatants were collected 18 h after infection and viral titers were determined by plaque assay. Data are mean ± SD of three independent assays performed in duplicate. Significant differences (one-way ANOVA, p values) are indicated
    Figure Legend Snippet: FMDV infection is enhanced by treatment with the gap-junction inhibitor carbenoxolone (CBX). IBRS-2 ( a ), SK6 ( b ) or WSL ( c ) cells (2 × 10 5 ) were treated with CBX at different concentrations for 3 h and then infected with FMDV O1K at an MOI of 0.01. Supernatants were collected 18 h after infection and viral titers were determined by plaque assay. Data are mean ± SD of three independent assays performed in duplicate. Significant differences (one-way ANOVA, p values) are indicated

    Techniques Used: Infection, Plaque Assay

    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
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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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    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)


    Bioz Verified Symbol ATCC is a verified supplier
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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).
    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, 2024-05
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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 cells  (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

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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
    https://www.bioz.com/result/swine kidney cell line pk 15/product/ATCC
    Average 99 stars, based on 1 article reviews
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    ATCC swine kidney cell lines sk6
    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 Lines Sk6, 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 lines sk6/product/ATCC
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    ATCC swine kidney cells
    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 Cells, 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
    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, 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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    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