bovine rotavirus ncdv g6p6  (ATCC)


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    Bovine rotavirus
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    Structured Review

    ATCC bovine rotavirus ncdv g6p6
    Inhibition of the RhoA/ROCK/MLC signaling pathway affects <t>rotavirus</t> infectivity and viral protein expression. MDCK cells were pretreated with non-cytotoxic concentrations of RhoA inhibitor (CT04), ROCK inhibitor (Y27632), or MLC inhibitor (blebbistatin) for 1 h at 37 °C and then infected with RVA strain DS-1 or <t>NCDV</t> (MOI = 10) for 12 h. ( a ) Total viral RNA was determined by real-time RT-PCR. ( b ) Virus titers were determined by cell culture immunofluorescence assay using cell lysates produced by three cycles of freezing and thawing; the results are expressed as fluorescent focus forming unit (FFU). ( c and d ) Viral VP6 protein was detected by western blot analysis. GAPDH was used as a loading control. The intensity of VP6 relative to GAPDH was determined by densitometric analysis and is indicated above each lane. All experiments were performed in triplicate. Representative images of different gels from each group are presented. Data are presented as the mean ± standard error of the mean from three independent experiments. Differences were evaluated using a one-way ANOVA. *p

    https://www.bioz.com/result/bovine rotavirus ncdv g6p6/product/ATCC
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    bovine rotavirus ncdv g6p6 - by Bioz Stars, 2021-09
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    1) Product Images from "Rotavirus-Induced Early Activation of the RhoA/ROCK/MLC Signaling Pathway Mediates the Disruption of Tight Junctions in Polarized MDCK Cells"

    Article Title: Rotavirus-Induced Early Activation of the RhoA/ROCK/MLC Signaling Pathway Mediates the Disruption of Tight Junctions in Polarized MDCK Cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-32352-y

    Inhibition of the RhoA/ROCK/MLC signaling pathway affects rotavirus infectivity and viral protein expression. MDCK cells were pretreated with non-cytotoxic concentrations of RhoA inhibitor (CT04), ROCK inhibitor (Y27632), or MLC inhibitor (blebbistatin) for 1 h at 37 °C and then infected with RVA strain DS-1 or NCDV (MOI = 10) for 12 h. ( a ) Total viral RNA was determined by real-time RT-PCR. ( b ) Virus titers were determined by cell culture immunofluorescence assay using cell lysates produced by three cycles of freezing and thawing; the results are expressed as fluorescent focus forming unit (FFU). ( c and d ) Viral VP6 protein was detected by western blot analysis. GAPDH was used as a loading control. The intensity of VP6 relative to GAPDH was determined by densitometric analysis and is indicated above each lane. All experiments were performed in triplicate. Representative images of different gels from each group are presented. Data are presented as the mean ± standard error of the mean from three independent experiments. Differences were evaluated using a one-way ANOVA. *p
    Figure Legend Snippet: Inhibition of the RhoA/ROCK/MLC signaling pathway affects rotavirus infectivity and viral protein expression. MDCK cells were pretreated with non-cytotoxic concentrations of RhoA inhibitor (CT04), ROCK inhibitor (Y27632), or MLC inhibitor (blebbistatin) for 1 h at 37 °C and then infected with RVA strain DS-1 or NCDV (MOI = 10) for 12 h. ( a ) Total viral RNA was determined by real-time RT-PCR. ( b ) Virus titers were determined by cell culture immunofluorescence assay using cell lysates produced by three cycles of freezing and thawing; the results are expressed as fluorescent focus forming unit (FFU). ( c and d ) Viral VP6 protein was detected by western blot analysis. GAPDH was used as a loading control. The intensity of VP6 relative to GAPDH was determined by densitometric analysis and is indicated above each lane. All experiments were performed in triplicate. Representative images of different gels from each group are presented. Data are presented as the mean ± standard error of the mean from three independent experiments. Differences were evaluated using a one-way ANOVA. *p

    Techniques Used: Inhibition, Infection, Expressing, Quantitative RT-PCR, Cell Culture, Immunofluorescence, Produced, Western Blot

    The RhoA/ROCK signaling pathway is involved in rotavirus-induced early activation of pMLC. ( a and b ) Confluent MDCK monolayers were mock-infected or infected with RVA strain ( a ) DS-1 or ( b ) NCDV (MOI = 10) for the indicated time points. The cells were then harvested and subjected to a Rhotekin pull-down assay to measure RhoA activation and then analyzed by western blot analysis using an anti-RhoA antibody or directly analyzed by western blot analysis to examine the expression levels of ROCK using specific primary antibody. ( c and d ) Confluent MDCK monolayers were pretreated with or without ( c ) RhoA inhibitor (CT04) or ( d ) ROCK inhibitor (Y27632) at the indicated doses for 1 h at 37 °C and then infected with strain DS-1 or NCDV (MOI = 10). Cell lysates were harvested at 1 h post-infection, and the expression levels of ROCK and/or pMLC were evaluated by western blot analysis. GAPDH was used as a loading control. The intensities of RhoA, ROCK, and pMLC relative to GAPDH were determined by densitometric analysis and are indicated above each lane. All experiments were performed in triplicate and representative images of different gels from each group are presented.
    Figure Legend Snippet: The RhoA/ROCK signaling pathway is involved in rotavirus-induced early activation of pMLC. ( a and b ) Confluent MDCK monolayers were mock-infected or infected with RVA strain ( a ) DS-1 or ( b ) NCDV (MOI = 10) for the indicated time points. The cells were then harvested and subjected to a Rhotekin pull-down assay to measure RhoA activation and then analyzed by western blot analysis using an anti-RhoA antibody or directly analyzed by western blot analysis to examine the expression levels of ROCK using specific primary antibody. ( c and d ) Confluent MDCK monolayers were pretreated with or without ( c ) RhoA inhibitor (CT04) or ( d ) ROCK inhibitor (Y27632) at the indicated doses for 1 h at 37 °C and then infected with strain DS-1 or NCDV (MOI = 10). Cell lysates were harvested at 1 h post-infection, and the expression levels of ROCK and/or pMLC were evaluated by western blot analysis. GAPDH was used as a loading control. The intensities of RhoA, ROCK, and pMLC relative to GAPDH were determined by densitometric analysis and are indicated above each lane. All experiments were performed in triplicate and representative images of different gels from each group are presented.

    Techniques Used: Activation Assay, Infection, Pull Down Assay, Western Blot, Expressing

    Inhibition of the RhoA/ROCK/MLC pathway restores the gate and fence functions of tight junction in rotavirus-infected polarized epithelial cells. MDCK monolayers grown on transwell filters were untreated or treated with the RhoA inhibitor (CT04), ROCK inhibitor (Y27632), or MLC inhibitor (blebbistatin) for 1 h at 37 °C and then mock-inoculated or inoculated with RVA strain ( a ) DS-1 or ( b ) NCDV (MOI = 10) for 5, 60, or 120 min. ( a and b ) Paracellular flux of 4-kDa FITC-dextran (FD4) and 70-kDa FITC dextran (FD70) was measured in the apical to basolateral direction. The amount of FITC dextran diffused to the basolateral side of the monolayer was normalized by the average obtained from control MDCK cells. As a positive control, MDCK monolayers were treated for 10 min with 1.8 mM EGTA. ( c and d ) Distribution of the membrane fluorescent marker BodipyFL-C12-sphingomyelin/BSA (5 nmol/ml) loaded onto the apical surface of MDCK cells was determined by z-sectioning using a confocal microscope. All experiments were performed in triplicate; c and d panels show representative results. Data are presented as the mean ± standard error of the mean from three independent experiments. Differences were evaluated using a one-way ANOVA. *p
    Figure Legend Snippet: Inhibition of the RhoA/ROCK/MLC pathway restores the gate and fence functions of tight junction in rotavirus-infected polarized epithelial cells. MDCK monolayers grown on transwell filters were untreated or treated with the RhoA inhibitor (CT04), ROCK inhibitor (Y27632), or MLC inhibitor (blebbistatin) for 1 h at 37 °C and then mock-inoculated or inoculated with RVA strain ( a ) DS-1 or ( b ) NCDV (MOI = 10) for 5, 60, or 120 min. ( a and b ) Paracellular flux of 4-kDa FITC-dextran (FD4) and 70-kDa FITC dextran (FD70) was measured in the apical to basolateral direction. The amount of FITC dextran diffused to the basolateral side of the monolayer was normalized by the average obtained from control MDCK cells. As a positive control, MDCK monolayers were treated for 10 min with 1.8 mM EGTA. ( c and d ) Distribution of the membrane fluorescent marker BodipyFL-C12-sphingomyelin/BSA (5 nmol/ml) loaded onto the apical surface of MDCK cells was determined by z-sectioning using a confocal microscope. All experiments were performed in triplicate; c and d panels show representative results. Data are presented as the mean ± standard error of the mean from three independent experiments. Differences were evaluated using a one-way ANOVA. *p

    Techniques Used: Inhibition, Infection, Positive Control, Marker, Microscopy

    Rotavirus VP8* protein triggers activation of the RhoA/ROCK/MLC pathway. ( a and b ) Confluent MDCK monolayers were incubated with recombinant GST-tagged VP8* protein of RVA strain ( a ) DS-1 or ( b ) NCDV (10 μg/ml) for the indicated time points. Cell lysates were subjected to a Rhotekin pull-down assay to measure RhoA activation and then analyzed by western blot analysis using an anti-RhoA antibody or directly analyzed by western blot analysis to check the expression level of ROCK, pMLC, and MLC2 using the relevant antibodies. GAPDH was used as a loading control. ( c and d ) Confluent MDCK monolayers were either mock-treated or pretreated with ( c ) RhoA inhibitor CT04 or ( d ) ROCK inhibitor Y27632 for 1 h at 37 °C and then incubated with the purified VP8* protein of RVA strain DS-1 or NCDV (10 μg/ml). Cell lysates were harvested at 1 h and the expression levels of ROCK and/or pMLC were evaluated by western blot analysis using the relevant antibodies. GAPDH was used as a loading control. All experiments were performed in triplicate and representative images of different gels from each group are presented. The intensities of RhoA, ROCK, and pMLC relative to GAPDH were determined by densitometric analysis and are indicated above each lane.
    Figure Legend Snippet: Rotavirus VP8* protein triggers activation of the RhoA/ROCK/MLC pathway. ( a and b ) Confluent MDCK monolayers were incubated with recombinant GST-tagged VP8* protein of RVA strain ( a ) DS-1 or ( b ) NCDV (10 μg/ml) for the indicated time points. Cell lysates were subjected to a Rhotekin pull-down assay to measure RhoA activation and then analyzed by western blot analysis using an anti-RhoA antibody or directly analyzed by western blot analysis to check the expression level of ROCK, pMLC, and MLC2 using the relevant antibodies. GAPDH was used as a loading control. ( c and d ) Confluent MDCK monolayers were either mock-treated or pretreated with ( c ) RhoA inhibitor CT04 or ( d ) ROCK inhibitor Y27632 for 1 h at 37 °C and then incubated with the purified VP8* protein of RVA strain DS-1 or NCDV (10 μg/ml). Cell lysates were harvested at 1 h and the expression levels of ROCK and/or pMLC were evaluated by western blot analysis using the relevant antibodies. GAPDH was used as a loading control. All experiments were performed in triplicate and representative images of different gels from each group are presented. The intensities of RhoA, ROCK, and pMLC relative to GAPDH were determined by densitometric analysis and are indicated above each lane.

    Techniques Used: Activation Assay, Incubation, Recombinant, Pull Down Assay, Western Blot, Expressing, Purification

    Inhibition of the RhoA/ROCK/MLC pathway restores tight junction resistance in rotavirus-infected polarized epithelial cells. ( a and b ) MDCK monolayers grown on transwell filters were pretreated with or without RhoA inhibitor (CT04), ROCK inhibitor (Y27632), or MLC inhibitor (blebbistatin) for 1 h at 37 °C and then mock-inoculated or inoculated with RVA strain ( a ) DS-1 or ( b ) NCDV (MOI = 10), followed by determination of TER at the indicated time points. Net TER was calculated by subtracting the background (membrane filter without cells) and multiplying the resistance (Ω) by the area (0.33 cm 2 ) of the filter. All experiments were performed in triplicate. Data are presented as the mean ± standard error of the mean from three independent experiments.
    Figure Legend Snippet: Inhibition of the RhoA/ROCK/MLC pathway restores tight junction resistance in rotavirus-infected polarized epithelial cells. ( a and b ) MDCK monolayers grown on transwell filters were pretreated with or without RhoA inhibitor (CT04), ROCK inhibitor (Y27632), or MLC inhibitor (blebbistatin) for 1 h at 37 °C and then mock-inoculated or inoculated with RVA strain ( a ) DS-1 or ( b ) NCDV (MOI = 10), followed by determination of TER at the indicated time points. Net TER was calculated by subtracting the background (membrane filter without cells) and multiplying the resistance (Ω) by the area (0.33 cm 2 ) of the filter. All experiments were performed in triplicate. Data are presented as the mean ± standard error of the mean from three independent experiments.

    Techniques Used: Inhibition, Infection

    Rotavirus infection-induced phosphorylation of MLC in polarized epithelial cells is an early event. ( a and b ) Human RVA DS-1 and bovine RVA NCDV strains (MOI = 10) were inoculated into confluent MDCK monolayers, and cells were harvested at the indicated time points. Cell lysates were subjected to western blot analysis to determine expression levels of phosphorylated MLC (pMLC) and MLC2 using the relevant antibodies. GAPDH was used as a loading control. The intensity of pMLC relative to GAPDH was determined by densitometric analysis and is indicated above each lane. All experiments were performed in triplicate and representative images of different gels from each group are presented.
    Figure Legend Snippet: Rotavirus infection-induced phosphorylation of MLC in polarized epithelial cells is an early event. ( a and b ) Human RVA DS-1 and bovine RVA NCDV strains (MOI = 10) were inoculated into confluent MDCK monolayers, and cells were harvested at the indicated time points. Cell lysates were subjected to western blot analysis to determine expression levels of phosphorylated MLC (pMLC) and MLC2 using the relevant antibodies. GAPDH was used as a loading control. The intensity of pMLC relative to GAPDH was determined by densitometric analysis and is indicated above each lane. All experiments were performed in triplicate and representative images of different gels from each group are presented.

    Techniques Used: Infection, Western Blot, Expressing

    Distribution of tight junction proteins is altered by the rotavirus-induced RhoA/ROCK/MLC signaling pathway. ( a – d ) MDCK monolayers were untreated or treated with RhoA inhibitor (CT04), ROCK inhibitor (Y27632), or MLC inhibitor (blebbistatin) for 1 h at 37 °C and then mock-inoculated or inoculated with RVA strain ( a and b ) DS-1 or ( c and d ) NCDV (MOI = 10) for 1 h. Cells were then fixed, permeabilized, and prepared for confocal microscopy using rabbit anti-ZO-1, occludin, claudin, and JAM-A antibodies and relevant secondary antibodies. All experiments were performed in triplicate and representative images are shown. The scale bars correspond to 20 μm. ( b and d ) Quantification of the internalization of each TJ molecule (shown as a percentage of intracellular fluorescence/total fluorescence) as described in Materials and Methods.
    Figure Legend Snippet: Distribution of tight junction proteins is altered by the rotavirus-induced RhoA/ROCK/MLC signaling pathway. ( a – d ) MDCK monolayers were untreated or treated with RhoA inhibitor (CT04), ROCK inhibitor (Y27632), or MLC inhibitor (blebbistatin) for 1 h at 37 °C and then mock-inoculated or inoculated with RVA strain ( a and b ) DS-1 or ( c and d ) NCDV (MOI = 10) for 1 h. Cells were then fixed, permeabilized, and prepared for confocal microscopy using rabbit anti-ZO-1, occludin, claudin, and JAM-A antibodies and relevant secondary antibodies. All experiments were performed in triplicate and representative images are shown. The scale bars correspond to 20 μm. ( b and d ) Quantification of the internalization of each TJ molecule (shown as a percentage of intracellular fluorescence/total fluorescence) as described in Materials and Methods.

    Techniques Used: Confocal Microscopy, Fluorescence

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    ATCC bovine rotavirus ncdv g6p6
    Inhibition of the RhoA/ROCK/MLC signaling pathway affects <t>rotavirus</t> infectivity and viral protein expression. MDCK cells were pretreated with non-cytotoxic concentrations of RhoA inhibitor (CT04), ROCK inhibitor (Y27632), or MLC inhibitor (blebbistatin) for 1 h at 37 °C and then infected with RVA strain DS-1 or <t>NCDV</t> (MOI = 10) for 12 h. ( a ) Total viral RNA was determined by real-time RT-PCR. ( b ) Virus titers were determined by cell culture immunofluorescence assay using cell lysates produced by three cycles of freezing and thawing; the results are expressed as fluorescent focus forming unit (FFU). ( c and d ) Viral VP6 protein was detected by western blot analysis. GAPDH was used as a loading control. The intensity of VP6 relative to GAPDH was determined by densitometric analysis and is indicated above each lane. All experiments were performed in triplicate. Representative images of different gels from each group are presented. Data are presented as the mean ± standard error of the mean from three independent experiments. Differences were evaluated using a one-way ANOVA. *p
    Bovine Rotavirus Ncdv G6p6, supplied by ATCC, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bovine rotavirus ncdv g6p6/product/ATCC
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    Inhibition of the RhoA/ROCK/MLC signaling pathway affects rotavirus infectivity and viral protein expression. MDCK cells were pretreated with non-cytotoxic concentrations of RhoA inhibitor (CT04), ROCK inhibitor (Y27632), or MLC inhibitor (blebbistatin) for 1 h at 37 °C and then infected with RVA strain DS-1 or NCDV (MOI = 10) for 12 h. ( a ) Total viral RNA was determined by real-time RT-PCR. ( b ) Virus titers were determined by cell culture immunofluorescence assay using cell lysates produced by three cycles of freezing and thawing; the results are expressed as fluorescent focus forming unit (FFU). ( c and d ) Viral VP6 protein was detected by western blot analysis. GAPDH was used as a loading control. The intensity of VP6 relative to GAPDH was determined by densitometric analysis and is indicated above each lane. All experiments were performed in triplicate. Representative images of different gels from each group are presented. Data are presented as the mean ± standard error of the mean from three independent experiments. Differences were evaluated using a one-way ANOVA. *p

    Journal: Scientific Reports

    Article Title: Rotavirus-Induced Early Activation of the RhoA/ROCK/MLC Signaling Pathway Mediates the Disruption of Tight Junctions in Polarized MDCK Cells

    doi: 10.1038/s41598-018-32352-y

    Figure Lengend Snippet: Inhibition of the RhoA/ROCK/MLC signaling pathway affects rotavirus infectivity and viral protein expression. MDCK cells were pretreated with non-cytotoxic concentrations of RhoA inhibitor (CT04), ROCK inhibitor (Y27632), or MLC inhibitor (blebbistatin) for 1 h at 37 °C and then infected with RVA strain DS-1 or NCDV (MOI = 10) for 12 h. ( a ) Total viral RNA was determined by real-time RT-PCR. ( b ) Virus titers were determined by cell culture immunofluorescence assay using cell lysates produced by three cycles of freezing and thawing; the results are expressed as fluorescent focus forming unit (FFU). ( c and d ) Viral VP6 protein was detected by western blot analysis. GAPDH was used as a loading control. The intensity of VP6 relative to GAPDH was determined by densitometric analysis and is indicated above each lane. All experiments were performed in triplicate. Representative images of different gels from each group are presented. Data are presented as the mean ± standard error of the mean from three independent experiments. Differences were evaluated using a one-way ANOVA. *p

    Article Snippet: The human rotavirus DS-1 (G2P1B[4]) and bovine rotavirus NCDV (G6P6[1]) strains purchased from ATCC were preactivated with 10 μg/ml crystalized trypsin (Cat. No. 27250-018; Gibco, Fort Worth, TX, USA) and propagated in MA104 cells as described previously .

    Techniques: Inhibition, Infection, Expressing, Quantitative RT-PCR, Cell Culture, Immunofluorescence, Produced, Western Blot

    The RhoA/ROCK signaling pathway is involved in rotavirus-induced early activation of pMLC. ( a and b ) Confluent MDCK monolayers were mock-infected or infected with RVA strain ( a ) DS-1 or ( b ) NCDV (MOI = 10) for the indicated time points. The cells were then harvested and subjected to a Rhotekin pull-down assay to measure RhoA activation and then analyzed by western blot analysis using an anti-RhoA antibody or directly analyzed by western blot analysis to examine the expression levels of ROCK using specific primary antibody. ( c and d ) Confluent MDCK monolayers were pretreated with or without ( c ) RhoA inhibitor (CT04) or ( d ) ROCK inhibitor (Y27632) at the indicated doses for 1 h at 37 °C and then infected with strain DS-1 or NCDV (MOI = 10). Cell lysates were harvested at 1 h post-infection, and the expression levels of ROCK and/or pMLC were evaluated by western blot analysis. GAPDH was used as a loading control. The intensities of RhoA, ROCK, and pMLC relative to GAPDH were determined by densitometric analysis and are indicated above each lane. All experiments were performed in triplicate and representative images of different gels from each group are presented.

    Journal: Scientific Reports

    Article Title: Rotavirus-Induced Early Activation of the RhoA/ROCK/MLC Signaling Pathway Mediates the Disruption of Tight Junctions in Polarized MDCK Cells

    doi: 10.1038/s41598-018-32352-y

    Figure Lengend Snippet: The RhoA/ROCK signaling pathway is involved in rotavirus-induced early activation of pMLC. ( a and b ) Confluent MDCK monolayers were mock-infected or infected with RVA strain ( a ) DS-1 or ( b ) NCDV (MOI = 10) for the indicated time points. The cells were then harvested and subjected to a Rhotekin pull-down assay to measure RhoA activation and then analyzed by western blot analysis using an anti-RhoA antibody or directly analyzed by western blot analysis to examine the expression levels of ROCK using specific primary antibody. ( c and d ) Confluent MDCK monolayers were pretreated with or without ( c ) RhoA inhibitor (CT04) or ( d ) ROCK inhibitor (Y27632) at the indicated doses for 1 h at 37 °C and then infected with strain DS-1 or NCDV (MOI = 10). Cell lysates were harvested at 1 h post-infection, and the expression levels of ROCK and/or pMLC were evaluated by western blot analysis. GAPDH was used as a loading control. The intensities of RhoA, ROCK, and pMLC relative to GAPDH were determined by densitometric analysis and are indicated above each lane. All experiments were performed in triplicate and representative images of different gels from each group are presented.

    Article Snippet: The human rotavirus DS-1 (G2P1B[4]) and bovine rotavirus NCDV (G6P6[1]) strains purchased from ATCC were preactivated with 10 μg/ml crystalized trypsin (Cat. No. 27250-018; Gibco, Fort Worth, TX, USA) and propagated in MA104 cells as described previously .

    Techniques: Activation Assay, Infection, Pull Down Assay, Western Blot, Expressing

    Inhibition of the RhoA/ROCK/MLC pathway restores the gate and fence functions of tight junction in rotavirus-infected polarized epithelial cells. MDCK monolayers grown on transwell filters were untreated or treated with the RhoA inhibitor (CT04), ROCK inhibitor (Y27632), or MLC inhibitor (blebbistatin) for 1 h at 37 °C and then mock-inoculated or inoculated with RVA strain ( a ) DS-1 or ( b ) NCDV (MOI = 10) for 5, 60, or 120 min. ( a and b ) Paracellular flux of 4-kDa FITC-dextran (FD4) and 70-kDa FITC dextran (FD70) was measured in the apical to basolateral direction. The amount of FITC dextran diffused to the basolateral side of the monolayer was normalized by the average obtained from control MDCK cells. As a positive control, MDCK monolayers were treated for 10 min with 1.8 mM EGTA. ( c and d ) Distribution of the membrane fluorescent marker BodipyFL-C12-sphingomyelin/BSA (5 nmol/ml) loaded onto the apical surface of MDCK cells was determined by z-sectioning using a confocal microscope. All experiments were performed in triplicate; c and d panels show representative results. Data are presented as the mean ± standard error of the mean from three independent experiments. Differences were evaluated using a one-way ANOVA. *p

    Journal: Scientific Reports

    Article Title: Rotavirus-Induced Early Activation of the RhoA/ROCK/MLC Signaling Pathway Mediates the Disruption of Tight Junctions in Polarized MDCK Cells

    doi: 10.1038/s41598-018-32352-y

    Figure Lengend Snippet: Inhibition of the RhoA/ROCK/MLC pathway restores the gate and fence functions of tight junction in rotavirus-infected polarized epithelial cells. MDCK monolayers grown on transwell filters were untreated or treated with the RhoA inhibitor (CT04), ROCK inhibitor (Y27632), or MLC inhibitor (blebbistatin) for 1 h at 37 °C and then mock-inoculated or inoculated with RVA strain ( a ) DS-1 or ( b ) NCDV (MOI = 10) for 5, 60, or 120 min. ( a and b ) Paracellular flux of 4-kDa FITC-dextran (FD4) and 70-kDa FITC dextran (FD70) was measured in the apical to basolateral direction. The amount of FITC dextran diffused to the basolateral side of the monolayer was normalized by the average obtained from control MDCK cells. As a positive control, MDCK monolayers were treated for 10 min with 1.8 mM EGTA. ( c and d ) Distribution of the membrane fluorescent marker BodipyFL-C12-sphingomyelin/BSA (5 nmol/ml) loaded onto the apical surface of MDCK cells was determined by z-sectioning using a confocal microscope. All experiments were performed in triplicate; c and d panels show representative results. Data are presented as the mean ± standard error of the mean from three independent experiments. Differences were evaluated using a one-way ANOVA. *p

    Article Snippet: The human rotavirus DS-1 (G2P1B[4]) and bovine rotavirus NCDV (G6P6[1]) strains purchased from ATCC were preactivated with 10 μg/ml crystalized trypsin (Cat. No. 27250-018; Gibco, Fort Worth, TX, USA) and propagated in MA104 cells as described previously .

    Techniques: Inhibition, Infection, Positive Control, Marker, Microscopy

    Rotavirus VP8* protein triggers activation of the RhoA/ROCK/MLC pathway. ( a and b ) Confluent MDCK monolayers were incubated with recombinant GST-tagged VP8* protein of RVA strain ( a ) DS-1 or ( b ) NCDV (10 μg/ml) for the indicated time points. Cell lysates were subjected to a Rhotekin pull-down assay to measure RhoA activation and then analyzed by western blot analysis using an anti-RhoA antibody or directly analyzed by western blot analysis to check the expression level of ROCK, pMLC, and MLC2 using the relevant antibodies. GAPDH was used as a loading control. ( c and d ) Confluent MDCK monolayers were either mock-treated or pretreated with ( c ) RhoA inhibitor CT04 or ( d ) ROCK inhibitor Y27632 for 1 h at 37 °C and then incubated with the purified VP8* protein of RVA strain DS-1 or NCDV (10 μg/ml). Cell lysates were harvested at 1 h and the expression levels of ROCK and/or pMLC were evaluated by western blot analysis using the relevant antibodies. GAPDH was used as a loading control. All experiments were performed in triplicate and representative images of different gels from each group are presented. The intensities of RhoA, ROCK, and pMLC relative to GAPDH were determined by densitometric analysis and are indicated above each lane.

    Journal: Scientific Reports

    Article Title: Rotavirus-Induced Early Activation of the RhoA/ROCK/MLC Signaling Pathway Mediates the Disruption of Tight Junctions in Polarized MDCK Cells

    doi: 10.1038/s41598-018-32352-y

    Figure Lengend Snippet: Rotavirus VP8* protein triggers activation of the RhoA/ROCK/MLC pathway. ( a and b ) Confluent MDCK monolayers were incubated with recombinant GST-tagged VP8* protein of RVA strain ( a ) DS-1 or ( b ) NCDV (10 μg/ml) for the indicated time points. Cell lysates were subjected to a Rhotekin pull-down assay to measure RhoA activation and then analyzed by western blot analysis using an anti-RhoA antibody or directly analyzed by western blot analysis to check the expression level of ROCK, pMLC, and MLC2 using the relevant antibodies. GAPDH was used as a loading control. ( c and d ) Confluent MDCK monolayers were either mock-treated or pretreated with ( c ) RhoA inhibitor CT04 or ( d ) ROCK inhibitor Y27632 for 1 h at 37 °C and then incubated with the purified VP8* protein of RVA strain DS-1 or NCDV (10 μg/ml). Cell lysates were harvested at 1 h and the expression levels of ROCK and/or pMLC were evaluated by western blot analysis using the relevant antibodies. GAPDH was used as a loading control. All experiments were performed in triplicate and representative images of different gels from each group are presented. The intensities of RhoA, ROCK, and pMLC relative to GAPDH were determined by densitometric analysis and are indicated above each lane.

    Article Snippet: The human rotavirus DS-1 (G2P1B[4]) and bovine rotavirus NCDV (G6P6[1]) strains purchased from ATCC were preactivated with 10 μg/ml crystalized trypsin (Cat. No. 27250-018; Gibco, Fort Worth, TX, USA) and propagated in MA104 cells as described previously .

    Techniques: Activation Assay, Incubation, Recombinant, Pull Down Assay, Western Blot, Expressing, Purification

    Inhibition of the RhoA/ROCK/MLC pathway restores tight junction resistance in rotavirus-infected polarized epithelial cells. ( a and b ) MDCK monolayers grown on transwell filters were pretreated with or without RhoA inhibitor (CT04), ROCK inhibitor (Y27632), or MLC inhibitor (blebbistatin) for 1 h at 37 °C and then mock-inoculated or inoculated with RVA strain ( a ) DS-1 or ( b ) NCDV (MOI = 10), followed by determination of TER at the indicated time points. Net TER was calculated by subtracting the background (membrane filter without cells) and multiplying the resistance (Ω) by the area (0.33 cm 2 ) of the filter. All experiments were performed in triplicate. Data are presented as the mean ± standard error of the mean from three independent experiments.

    Journal: Scientific Reports

    Article Title: Rotavirus-Induced Early Activation of the RhoA/ROCK/MLC Signaling Pathway Mediates the Disruption of Tight Junctions in Polarized MDCK Cells

    doi: 10.1038/s41598-018-32352-y

    Figure Lengend Snippet: Inhibition of the RhoA/ROCK/MLC pathway restores tight junction resistance in rotavirus-infected polarized epithelial cells. ( a and b ) MDCK monolayers grown on transwell filters were pretreated with or without RhoA inhibitor (CT04), ROCK inhibitor (Y27632), or MLC inhibitor (blebbistatin) for 1 h at 37 °C and then mock-inoculated or inoculated with RVA strain ( a ) DS-1 or ( b ) NCDV (MOI = 10), followed by determination of TER at the indicated time points. Net TER was calculated by subtracting the background (membrane filter without cells) and multiplying the resistance (Ω) by the area (0.33 cm 2 ) of the filter. All experiments were performed in triplicate. Data are presented as the mean ± standard error of the mean from three independent experiments.

    Article Snippet: The human rotavirus DS-1 (G2P1B[4]) and bovine rotavirus NCDV (G6P6[1]) strains purchased from ATCC were preactivated with 10 μg/ml crystalized trypsin (Cat. No. 27250-018; Gibco, Fort Worth, TX, USA) and propagated in MA104 cells as described previously .

    Techniques: Inhibition, Infection

    Rotavirus infection-induced phosphorylation of MLC in polarized epithelial cells is an early event. ( a and b ) Human RVA DS-1 and bovine RVA NCDV strains (MOI = 10) were inoculated into confluent MDCK monolayers, and cells were harvested at the indicated time points. Cell lysates were subjected to western blot analysis to determine expression levels of phosphorylated MLC (pMLC) and MLC2 using the relevant antibodies. GAPDH was used as a loading control. The intensity of pMLC relative to GAPDH was determined by densitometric analysis and is indicated above each lane. All experiments were performed in triplicate and representative images of different gels from each group are presented.

    Journal: Scientific Reports

    Article Title: Rotavirus-Induced Early Activation of the RhoA/ROCK/MLC Signaling Pathway Mediates the Disruption of Tight Junctions in Polarized MDCK Cells

    doi: 10.1038/s41598-018-32352-y

    Figure Lengend Snippet: Rotavirus infection-induced phosphorylation of MLC in polarized epithelial cells is an early event. ( a and b ) Human RVA DS-1 and bovine RVA NCDV strains (MOI = 10) were inoculated into confluent MDCK monolayers, and cells were harvested at the indicated time points. Cell lysates were subjected to western blot analysis to determine expression levels of phosphorylated MLC (pMLC) and MLC2 using the relevant antibodies. GAPDH was used as a loading control. The intensity of pMLC relative to GAPDH was determined by densitometric analysis and is indicated above each lane. All experiments were performed in triplicate and representative images of different gels from each group are presented.

    Article Snippet: The human rotavirus DS-1 (G2P1B[4]) and bovine rotavirus NCDV (G6P6[1]) strains purchased from ATCC were preactivated with 10 μg/ml crystalized trypsin (Cat. No. 27250-018; Gibco, Fort Worth, TX, USA) and propagated in MA104 cells as described previously .

    Techniques: Infection, Western Blot, Expressing

    Distribution of tight junction proteins is altered by the rotavirus-induced RhoA/ROCK/MLC signaling pathway. ( a – d ) MDCK monolayers were untreated or treated with RhoA inhibitor (CT04), ROCK inhibitor (Y27632), or MLC inhibitor (blebbistatin) for 1 h at 37 °C and then mock-inoculated or inoculated with RVA strain ( a and b ) DS-1 or ( c and d ) NCDV (MOI = 10) for 1 h. Cells were then fixed, permeabilized, and prepared for confocal microscopy using rabbit anti-ZO-1, occludin, claudin, and JAM-A antibodies and relevant secondary antibodies. All experiments were performed in triplicate and representative images are shown. The scale bars correspond to 20 μm. ( b and d ) Quantification of the internalization of each TJ molecule (shown as a percentage of intracellular fluorescence/total fluorescence) as described in Materials and Methods.

    Journal: Scientific Reports

    Article Title: Rotavirus-Induced Early Activation of the RhoA/ROCK/MLC Signaling Pathway Mediates the Disruption of Tight Junctions in Polarized MDCK Cells

    doi: 10.1038/s41598-018-32352-y

    Figure Lengend Snippet: Distribution of tight junction proteins is altered by the rotavirus-induced RhoA/ROCK/MLC signaling pathway. ( a – d ) MDCK monolayers were untreated or treated with RhoA inhibitor (CT04), ROCK inhibitor (Y27632), or MLC inhibitor (blebbistatin) for 1 h at 37 °C and then mock-inoculated or inoculated with RVA strain ( a and b ) DS-1 or ( c and d ) NCDV (MOI = 10) for 1 h. Cells were then fixed, permeabilized, and prepared for confocal microscopy using rabbit anti-ZO-1, occludin, claudin, and JAM-A antibodies and relevant secondary antibodies. All experiments were performed in triplicate and representative images are shown. The scale bars correspond to 20 μm. ( b and d ) Quantification of the internalization of each TJ molecule (shown as a percentage of intracellular fluorescence/total fluorescence) as described in Materials and Methods.

    Article Snippet: The human rotavirus DS-1 (G2P1B[4]) and bovine rotavirus NCDV (G6P6[1]) strains purchased from ATCC were preactivated with 10 μg/ml crystalized trypsin (Cat. No. 27250-018; Gibco, Fort Worth, TX, USA) and propagated in MA104 cells as described previously .

    Techniques: Confocal Microscopy, Fluorescence