hep 2 cells  (ATCC)


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    Structured Review

    ATCC hep 2 cells
    ICP22-dependent phosphorylation of RNA POL II CTD requires the US3 protein kinase. (A and D) <t>HEp-2</t> cells were mock infected or exposed to HSV-1(F), R325 (Δα22), R7356 (ΔU L 13), R7041 (ΔU S 3), or R7353 (ΔU L 13/ΔU
    Hep 2 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 78/100, based on 0 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hep 2 cells/product/ATCC
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    Images

    1) Product Images from "The Carboxyl-Terminal Domain of RNA Polymerase II Is Phosphorylated by a Complex Containing cdk9 and Infected-Cell Protein 22 of Herpes Simplex Virus 1"

    Article Title: The Carboxyl-Terminal Domain of RNA Polymerase II Is Phosphorylated by a Complex Containing cdk9 and Infected-Cell Protein 22 of Herpes Simplex Virus 1

    Journal:

    doi: 10.1128/JVI.79.11.6757-6762.2005

    ICP22-dependent phosphorylation of RNA POL II CTD requires the US3 protein kinase. (A and D) HEp-2 cells were mock infected or exposed to HSV-1(F), R325 (Δα22), R7356 (ΔU L 13), R7041 (ΔU S 3), or R7353 (ΔU L 13/ΔU
    Figure Legend Snippet: ICP22-dependent phosphorylation of RNA POL II CTD requires the US3 protein kinase. (A and D) HEp-2 cells were mock infected or exposed to HSV-1(F), R325 (Δα22), R7356 (ΔU L 13), R7041 (ΔU S 3), or R7353 (ΔU L 13/ΔU

    Techniques Used: Infection

    GST-cdk9 interacts with ICP22. Lysates of HEp-2 cells mock infected or infected with HSV-1(F) or R325 (ΔICP22) and incubated for 6 h were reacted with GST-cdk9. The bound proteins were solubilized, subjected to electrophoresis on a denaturing
    Figure Legend Snippet: GST-cdk9 interacts with ICP22. Lysates of HEp-2 cells mock infected or infected with HSV-1(F) or R325 (ΔICP22) and incubated for 6 h were reacted with GST-cdk9. The bound proteins were solubilized, subjected to electrophoresis on a denaturing

    Techniques Used: Infection, Incubation, Electrophoresis

    The interaction of GST-cdk9 chimeric protein with the 69,000- M r protein is dependent on ICP22 and the U S 3 protein kinase. Panel A: Autoradiogram of electrophoretically separated lysates of [ 35 S]Met-labeled HEp-2 cell proteins. The cells were harvested
    Figure Legend Snippet: The interaction of GST-cdk9 chimeric protein with the 69,000- M r protein is dependent on ICP22 and the U S 3 protein kinase. Panel A: Autoradiogram of electrophoretically separated lysates of [ 35 S]Met-labeled HEp-2 cell proteins. The cells were harvested

    Techniques Used: Labeling

    2) Product Images from "Translocation and Colocalization of ICP4 and ICP0 in Cells Infected with Herpes Simplex Virus 1 Mutants Lacking Glycoprotein E, Glycoprotein I, or the Virion Host Shutoff Product of the UL41 Gene ▿"

    Article Title: Translocation and Colocalization of ICP4 and ICP0 in Cells Infected with Herpes Simplex Virus 1 Mutants Lacking Glycoprotein E, Glycoprotein I, or the Virion Host Shutoff Product of the UL41 Gene ▿

    Journal:

    doi: 10.1128/JVI.02157-07

    Properties of the ΔgE, ΔgI, and ΔU L 41 viruses. (A and B) Expression patterns of gE and gI in ΔU L 41 virus-infected cells. HEp-2 cells were either mock infected or exposed (10 PFU/cell) to HSV-1(F), ΔU L 41, ΔgE-2
    Figure Legend Snippet: Properties of the ΔgE, ΔgI, and ΔU L 41 viruses. (A and B) Expression patterns of gE and gI in ΔU L 41 virus-infected cells. HEp-2 cells were either mock infected or exposed (10 PFU/cell) to HSV-1(F), ΔU L 41, ΔgE-2

    Techniques Used: Expressing, Infection

    Exportation of ICP4 from the nucleus and aggregation in the cytoplasm of the ΔgE and ΔU L 41 virus-infected cells. Hep-2 cells, seeded in four-well slides, were either mock infected or exposed (10 PFU/cell) to HSV-1(F), ΔgE, or ΔU
    Figure Legend Snippet: Exportation of ICP4 from the nucleus and aggregation in the cytoplasm of the ΔgE and ΔU L 41 virus-infected cells. Hep-2 cells, seeded in four-well slides, were either mock infected or exposed (10 PFU/cell) to HSV-1(F), ΔgE, or ΔU

    Techniques Used: Infection

    Electron photomicrographs of ICP0-containing structures in HSV-1(F)- and ΔgE-infected cells. HEp-2 cells were harvested at 18 h after infection with 10 PFU/cell of HSV-1(F) or the ΔgE mutant and processed for immunoelectron microscopy,
    Figure Legend Snippet: Electron photomicrographs of ICP0-containing structures in HSV-1(F)- and ΔgE-infected cells. HEp-2 cells were harvested at 18 h after infection with 10 PFU/cell of HSV-1(F) or the ΔgE mutant and processed for immunoelectron microscopy,

    Techniques Used: Infection, Mutagenesis, Immuno-Electron Microscopy

    3) Product Images from "The Transmembrane Domain of the Respiratory Syncytial Virus F Protein Is an Orientation-Independent Apical Plasma Membrane Sorting Sequence"

    Article Title: The Transmembrane Domain of the Respiratory Syncytial Virus F Protein Is an Orientation-Independent Apical Plasma Membrane Sorting Sequence

    Journal:

    doi: 10.1128/JVI.79.19.12528-12535.2005

    Cellular distribution and orientation of GFP-RSV F tail in nonpolarized epithelial cells. (a) HEp-2 cells were infected with RSV for 48 h to determine the cellular distribution of the F protein. RSV F (red) was distributed in a perinuclear manner, consistent
    Figure Legend Snippet: Cellular distribution and orientation of GFP-RSV F tail in nonpolarized epithelial cells. (a) HEp-2 cells were infected with RSV for 48 h to determine the cellular distribution of the F protein. RSV F (red) was distributed in a perinuclear manner, consistent

    Techniques Used: Infection

    ER and Golgi colocalization of GFP-RSV F tail constructs in nonpolarized HEp-2 cells. HEp-2 cells were transfected with GFP-RSV F tail (a and b) or GFP-RSV F tail (ΔCT) (c and d) to assess the codistribution of each protein with resident ER (a and
    Figure Legend Snippet: ER and Golgi colocalization of GFP-RSV F tail constructs in nonpolarized HEp-2 cells. HEp-2 cells were transfected with GFP-RSV F tail (a and b) or GFP-RSV F tail (ΔCT) (c and d) to assess the codistribution of each protein with resident ER (a and

    Techniques Used: Construct, Transfection

    Cellular distribution of RSV F opt in nonpolarized epithelial cells. (a) Schematic representation of full-length and mutant forms of RSV F opt . (b to e) HEp-2 cells were transfected with each RSV F opt construct noted in panel a to assess the cellular distribution
    Figure Legend Snippet: Cellular distribution of RSV F opt in nonpolarized epithelial cells. (a) Schematic representation of full-length and mutant forms of RSV F opt . (b to e) HEp-2 cells were transfected with each RSV F opt construct noted in panel a to assess the cellular distribution

    Techniques Used: Mutagenesis, Transfection, Construct

    Related Articles

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    Article Snippet: HEp-2 cells, obtained from the American Type Culture Collection (Rockville, MD), and telomerase-transformed human embryonic lung (HEL) fibroblasts, obtained from T. Shenk (Princeton), were grown in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum.

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    ATCC eagle s minimum essential medium
    Eagle S Minimum Essential Medium, supplied by ATCC, used in various techniques. Bioz Stars score: 90/100, based on 255 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC human epithelial hep 2
    Procaspase-3 is directly cleaved by EspC. (A) The presence of a caspase inhibitor plus a mutation in the serine protease motif causes a dramatic decrease in caspase-3 activation. <t>HEp-2</t> cells were pretreated with 50 µM z-VAD-fmk or only DMSO for 1 h. Cells were infected with the EPEC WT, Δ espC , or Δ espC /p espC S256I strain at an MOI of 10 for 4 h. Infected cells were lysed, and proteins were analyzed by immunoblotting using anti-caspase-3 and anti-β-actin as primary antibodies and HRP-conjugated anti-isotype secondary antibody. Data are expressed as the mean ± SEM from at least 3 independent experiments. Statistical analysis was performed using two-way ANOVA followed by Bonferroni’s multiple comparison test (**, P
    Human Epithelial Hep 2, supplied by ATCC, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    tu212  (ATCC)
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    ATCC tu212
    Overexpressed hsa-miR-138-2-3p activated JNK1/MAPK pathway. The vertical and horizontal axis stand for Gary value and treaments, respectively. It suggested that the expression of JNK1 between 100nM-TR and 100nMN-CR of Hep-2 (A), M2e (B) and <t>TU212</t> (C) CSCs were significant difference (** P
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    Image Search Results


    Procaspase-3 is directly cleaved by EspC. (A) The presence of a caspase inhibitor plus a mutation in the serine protease motif causes a dramatic decrease in caspase-3 activation. HEp-2 cells were pretreated with 50 µM z-VAD-fmk or only DMSO for 1 h. Cells were infected with the EPEC WT, Δ espC , or Δ espC /p espC S256I strain at an MOI of 10 for 4 h. Infected cells were lysed, and proteins were analyzed by immunoblotting using anti-caspase-3 and anti-β-actin as primary antibodies and HRP-conjugated anti-isotype secondary antibody. Data are expressed as the mean ± SEM from at least 3 independent experiments. Statistical analysis was performed using two-way ANOVA followed by Bonferroni’s multiple comparison test (**, P

    Journal: mBio

    Article Title: EspC, an Autotransporter Protein Secreted by Enteropathogenic Escherichia coli, Causes Apoptosis and Necrosis through Caspase and Calpain Activation, Including Direct Procaspase-3 Cleavage

    doi: 10.1128/mBio.00479-16

    Figure Lengend Snippet: Procaspase-3 is directly cleaved by EspC. (A) The presence of a caspase inhibitor plus a mutation in the serine protease motif causes a dramatic decrease in caspase-3 activation. HEp-2 cells were pretreated with 50 µM z-VAD-fmk or only DMSO for 1 h. Cells were infected with the EPEC WT, Δ espC , or Δ espC /p espC S256I strain at an MOI of 10 for 4 h. Infected cells were lysed, and proteins were analyzed by immunoblotting using anti-caspase-3 and anti-β-actin as primary antibodies and HRP-conjugated anti-isotype secondary antibody. Data are expressed as the mean ± SEM from at least 3 independent experiments. Statistical analysis was performed using two-way ANOVA followed by Bonferroni’s multiple comparison test (**, P

    Article Snippet: The human epithelial HEp-2 (ATCC CCL23) or HT-29 (ATCC HTB-38) cell lines were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT), 1% nonessential amino acids, 5 mM l -glutamine, penicillin (100 U/ml), and streptomycin (100 µg/ml).

    Techniques: Mutagenesis, Activation Assay, Infection

    EspC also induces caspase-independent cell death. (A and C) Caspase inhibition does not block apoptosis induced by EPEC. HEp-2 cells were pretreated with 50 µM z-VAD-fmk or only DMSO for 1 h. Cells were infected with the EPEC WT, Δ espC , or Δ escN strain at an MOI of 10 for 4 h (A) or treated with 100 µM cisplatin (CDDP) for 18 h (C). Mock-infected cells were used as a negative control. Cells were harvested and labeled with annexin V and analyzed by flow cytometry. Annexin V-positive cells were considered apoptotic cells. (B and D) Caspase inhibition blocks caspase-3 activity induced by EPEC. Activity of caspase-3 was determined in HEp-2 cells pretreated with 50 µM z-VAD-fmk or only DMSO for 1 h and infected with the EPEC WT or Δ espC strain at an MOI of 10 for 4 h (B) or 100 µM CDDP for 18 h (D). The whole-cell lysates were subjected to the caspase-3 activity assay as described in Materials and Methods. Activity is represented as fold change relative to uninfected cells. Data are expressed as the mean ± SEM from at least 3 independent experiments. Statistical analysis was performed using two-way ANOVA followed by Bonferroni’s multiple comparison test (*, P

    Journal: mBio

    Article Title: EspC, an Autotransporter Protein Secreted by Enteropathogenic Escherichia coli, Causes Apoptosis and Necrosis through Caspase and Calpain Activation, Including Direct Procaspase-3 Cleavage

    doi: 10.1128/mBio.00479-16

    Figure Lengend Snippet: EspC also induces caspase-independent cell death. (A and C) Caspase inhibition does not block apoptosis induced by EPEC. HEp-2 cells were pretreated with 50 µM z-VAD-fmk or only DMSO for 1 h. Cells were infected with the EPEC WT, Δ espC , or Δ escN strain at an MOI of 10 for 4 h (A) or treated with 100 µM cisplatin (CDDP) for 18 h (C). Mock-infected cells were used as a negative control. Cells were harvested and labeled with annexin V and analyzed by flow cytometry. Annexin V-positive cells were considered apoptotic cells. (B and D) Caspase inhibition blocks caspase-3 activity induced by EPEC. Activity of caspase-3 was determined in HEp-2 cells pretreated with 50 µM z-VAD-fmk or only DMSO for 1 h and infected with the EPEC WT or Δ espC strain at an MOI of 10 for 4 h (B) or 100 µM CDDP for 18 h (D). The whole-cell lysates were subjected to the caspase-3 activity assay as described in Materials and Methods. Activity is represented as fold change relative to uninfected cells. Data are expressed as the mean ± SEM from at least 3 independent experiments. Statistical analysis was performed using two-way ANOVA followed by Bonferroni’s multiple comparison test (*, P

    Article Snippet: The human epithelial HEp-2 (ATCC CCL23) or HT-29 (ATCC HTB-38) cell lines were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT), 1% nonessential amino acids, 5 mM l -glutamine, penicillin (100 U/ml), and streptomycin (100 µg/ml).

    Techniques: Inhibition, Blocking Assay, Infection, Negative Control, Labeling, Flow Cytometry, Cytometry, Activity Assay, Caspase-3 Activity Assay

    EspC induces depolarization of mitochondrial inner membrane (ΔΨ m ). (A) EspC is involved in the loss of mitochondrial membrane potential. HEp-2 cells were prestained with rhodamine 123 and then infected with bacterial strains as indicated at an MOI of 10 for 4 h. Purified EspC, EspC S256I , or EspC (900 nM) preincubated with PMSF was added during the infection to complement the double mutant as indicated. Staurosporine (STS) and Triton X-100 were used as positive controls. All cells were analyzed for the loss of mitochondrial inner membrane potential (ΔΨ m ) by flow cytometry. (B) Complementation of the EPEC Δ espC Δ espF strain with exogenous EspC. HEp-2 cells were infected with bacterial strains as indicated and supplemented with increasing molarities of purified EspC at an MOI of 10 for 3 h. Cells were processed and analyzed as indicated above. Data are expressed as the mean ± SEM from at least 3 independent experiments. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparison test for comparison to the (A) WT or (B) Δ espC Δ espF strain (*, P

    Journal: mBio

    Article Title: EspC, an Autotransporter Protein Secreted by Enteropathogenic Escherichia coli, Causes Apoptosis and Necrosis through Caspase and Calpain Activation, Including Direct Procaspase-3 Cleavage

    doi: 10.1128/mBio.00479-16

    Figure Lengend Snippet: EspC induces depolarization of mitochondrial inner membrane (ΔΨ m ). (A) EspC is involved in the loss of mitochondrial membrane potential. HEp-2 cells were prestained with rhodamine 123 and then infected with bacterial strains as indicated at an MOI of 10 for 4 h. Purified EspC, EspC S256I , or EspC (900 nM) preincubated with PMSF was added during the infection to complement the double mutant as indicated. Staurosporine (STS) and Triton X-100 were used as positive controls. All cells were analyzed for the loss of mitochondrial inner membrane potential (ΔΨ m ) by flow cytometry. (B) Complementation of the EPEC Δ espC Δ espF strain with exogenous EspC. HEp-2 cells were infected with bacterial strains as indicated and supplemented with increasing molarities of purified EspC at an MOI of 10 for 3 h. Cells were processed and analyzed as indicated above. Data are expressed as the mean ± SEM from at least 3 independent experiments. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparison test for comparison to the (A) WT or (B) Δ espC Δ espF strain (*, P

    Article Snippet: The human epithelial HEp-2 (ATCC CCL23) or HT-29 (ATCC HTB-38) cell lines were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT), 1% nonessential amino acids, 5 mM l -glutamine, penicillin (100 U/ml), and streptomycin (100 µg/ml).

    Techniques: Infection, Purification, Mutagenesis, Flow Cytometry, Cytometry

    EspC induces apoptosis and necrosis on epithelial cells. (A) Analyses of apoptotic and necrotic cells during kinetics of infection. FACS analysis via annexin V and PI staining was used to observe the induction of apoptosis and necrosis of HEp-2 cells infected by the EPEC WT, Δ espC , Δ espC /p espC , Δ espC /p espC S256I , or Δ escN strain at an MOI of 10 for the indicated lengths of time. Mock-infected cells were used as a negative control. Annexin V-negative and PI-negative cells represent live cells. Annexin V-positive and PI-negative cells represent the early apoptotic populations. Annexin V-positive and PI-positive cells represent the late apoptotic populations. Annexin V-negative and PI-positive cells represent the necrotic populations. Representative flow cytometric dot plots are shown. (B) Distribution of the infected cell populations at 4 h of infection. HEp-2 cells were infected with the bacterial strains for 4 h as indicated. Mock-infected cells were used as a negative control. Staurosporine (STS) at 1 µM and 0.1% Triton X-100 were used as positive controls for apoptosis or necrosis, respectively. The results were plotted and are shown as the mean ± SEM from at least 3 independent experiments.

    Journal: mBio

    Article Title: EspC, an Autotransporter Protein Secreted by Enteropathogenic Escherichia coli, Causes Apoptosis and Necrosis through Caspase and Calpain Activation, Including Direct Procaspase-3 Cleavage

    doi: 10.1128/mBio.00479-16

    Figure Lengend Snippet: EspC induces apoptosis and necrosis on epithelial cells. (A) Analyses of apoptotic and necrotic cells during kinetics of infection. FACS analysis via annexin V and PI staining was used to observe the induction of apoptosis and necrosis of HEp-2 cells infected by the EPEC WT, Δ espC , Δ espC /p espC , Δ espC /p espC S256I , or Δ escN strain at an MOI of 10 for the indicated lengths of time. Mock-infected cells were used as a negative control. Annexin V-negative and PI-negative cells represent live cells. Annexin V-positive and PI-negative cells represent the early apoptotic populations. Annexin V-positive and PI-positive cells represent the late apoptotic populations. Annexin V-negative and PI-positive cells represent the necrotic populations. Representative flow cytometric dot plots are shown. (B) Distribution of the infected cell populations at 4 h of infection. HEp-2 cells were infected with the bacterial strains for 4 h as indicated. Mock-infected cells were used as a negative control. Staurosporine (STS) at 1 µM and 0.1% Triton X-100 were used as positive controls for apoptosis or necrosis, respectively. The results were plotted and are shown as the mean ± SEM from at least 3 independent experiments.

    Article Snippet: The human epithelial HEp-2 (ATCC CCL23) or HT-29 (ATCC HTB-38) cell lines were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT), 1% nonessential amino acids, 5 mM l -glutamine, penicillin (100 U/ml), and streptomycin (100 µg/ml).

    Techniques: Infection, FACS, Staining, Negative Control, Flow Cytometry

    EspC is required for caspase-3 activation and activity. (A) EspC-producing EPEC induces procaspase-3 cleavage. HEp-2 cells were infected with the indicated bacterial strains at an MOI of 10 at the different times indicated. Infected cells were lysed, and proteins were analyzed by immunoblotting using anti-caspase-3, anti-β-actin, and anti-EspC as primary antibodies and HRP-conjugated anti-isotype secondary antibody. Samples were normalized using the maximum densitometric value. (B) Induction of procaspase-3 cleavage is complemented in the mutant strains by either espC or espF , and this effect is higher by espC complementation. Activation of caspase-3 was performed as indicated above, in cells infected by the indicated strains at 4 h of infection. (C) EspC-producing EPEC induces caspase-3 activity. Activity of caspase-3 was determined in HEp-2 cells infected by the EPEC WT, Δ espC , Δ espC /p espC , or Δ escN strain at an MOI of 10 for 4 h. The whole-cell lysates were subjected to the caspase activity assay by using a synthetic substrate as described in Materials and Methods. Activity is represented as fold change relative to uninfected cells. Data are shown as the mean ± SEM from at least 3 independent experiments. Statistical analyses were performed using (A) unpaired t test (*, P

    Journal: mBio

    Article Title: EspC, an Autotransporter Protein Secreted by Enteropathogenic Escherichia coli, Causes Apoptosis and Necrosis through Caspase and Calpain Activation, Including Direct Procaspase-3 Cleavage

    doi: 10.1128/mBio.00479-16

    Figure Lengend Snippet: EspC is required for caspase-3 activation and activity. (A) EspC-producing EPEC induces procaspase-3 cleavage. HEp-2 cells were infected with the indicated bacterial strains at an MOI of 10 at the different times indicated. Infected cells were lysed, and proteins were analyzed by immunoblotting using anti-caspase-3, anti-β-actin, and anti-EspC as primary antibodies and HRP-conjugated anti-isotype secondary antibody. Samples were normalized using the maximum densitometric value. (B) Induction of procaspase-3 cleavage is complemented in the mutant strains by either espC or espF , and this effect is higher by espC complementation. Activation of caspase-3 was performed as indicated above, in cells infected by the indicated strains at 4 h of infection. (C) EspC-producing EPEC induces caspase-3 activity. Activity of caspase-3 was determined in HEp-2 cells infected by the EPEC WT, Δ espC , Δ espC /p espC , or Δ escN strain at an MOI of 10 for 4 h. The whole-cell lysates were subjected to the caspase activity assay by using a synthetic substrate as described in Materials and Methods. Activity is represented as fold change relative to uninfected cells. Data are shown as the mean ± SEM from at least 3 independent experiments. Statistical analyses were performed using (A) unpaired t test (*, P

    Article Snippet: The human epithelial HEp-2 (ATCC CCL23) or HT-29 (ATCC HTB-38) cell lines were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT), 1% nonessential amino acids, 5 mM l -glutamine, penicillin (100 U/ml), and streptomycin (100 µg/ml).

    Techniques: Activation Assay, Activity Assay, Infection, Mutagenesis, Caspase Activity Assay

    EspC induces necrosis through calpain activation. (A) Kinetics of necrosis induction by the EPEC WT and Δ espC mutant and their inhibition by a calpain inhibitor. HEp-2 cells were pretreated with 50 µM calpain inhibitor I for 1 h or untreated. Cells were infected with either the EPEC WT or Δ espC mutant at an MOI of 10 for the indicated times. Cells were harvested, labeled with annexin V/PI, and analyzed by flow cytometry. Annexin V-negative and PI-positive cells were considered necrotic cells. (B) Comparison of cells infected with the different bacterial strains for 4 h. Cells were infected with the indicated strains, processed, and analyzed as indicated above. Data are shown as the mean ± SEM from at least 3 independent experiments. Statistical analyses were performed using (A) unpaired t test (**, P

    Journal: mBio

    Article Title: EspC, an Autotransporter Protein Secreted by Enteropathogenic Escherichia coli, Causes Apoptosis and Necrosis through Caspase and Calpain Activation, Including Direct Procaspase-3 Cleavage

    doi: 10.1128/mBio.00479-16

    Figure Lengend Snippet: EspC induces necrosis through calpain activation. (A) Kinetics of necrosis induction by the EPEC WT and Δ espC mutant and their inhibition by a calpain inhibitor. HEp-2 cells were pretreated with 50 µM calpain inhibitor I for 1 h or untreated. Cells were infected with either the EPEC WT or Δ espC mutant at an MOI of 10 for the indicated times. Cells were harvested, labeled with annexin V/PI, and analyzed by flow cytometry. Annexin V-negative and PI-positive cells were considered necrotic cells. (B) Comparison of cells infected with the different bacterial strains for 4 h. Cells were infected with the indicated strains, processed, and analyzed as indicated above. Data are shown as the mean ± SEM from at least 3 independent experiments. Statistical analyses were performed using (A) unpaired t test (**, P

    Article Snippet: The human epithelial HEp-2 (ATCC CCL23) or HT-29 (ATCC HTB-38) cell lines were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT), 1% nonessential amino acids, 5 mM l -glutamine, penicillin (100 U/ml), and streptomycin (100 µg/ml).

    Techniques: Activation Assay, Mutagenesis, Inhibition, Infection, Labeling, Flow Cytometry, Cytometry

    EspC stimulates Bax translocation to mitochondria and cytochrome c release. EspC induces Bax translocation at 4 h of infection (A) and release of cytochrome c from mitochondria to cytosol (B). HEp-2 cells were infected with the different strains as indicated at an MOI of 10 and for the indicated lengths of time. Host cell cytosolic and mitochondrial fractions were separated and analyzed by immunoblotting using (A) anti-Bax or (B) anti-cytochrome c as primary antibodies and HRP-conjugated anti-isotype secondary antibody. Blots were also probed for COX IV as a control marker for the mitochondrial fractions and GAPDH as a control marker for the cytosolic fractions. The blots shown are representative of three independent experiments. Asterisks indicate a clear increase induced by EPEC.

    Journal: mBio

    Article Title: EspC, an Autotransporter Protein Secreted by Enteropathogenic Escherichia coli, Causes Apoptosis and Necrosis through Caspase and Calpain Activation, Including Direct Procaspase-3 Cleavage

    doi: 10.1128/mBio.00479-16

    Figure Lengend Snippet: EspC stimulates Bax translocation to mitochondria and cytochrome c release. EspC induces Bax translocation at 4 h of infection (A) and release of cytochrome c from mitochondria to cytosol (B). HEp-2 cells were infected with the different strains as indicated at an MOI of 10 and for the indicated lengths of time. Host cell cytosolic and mitochondrial fractions were separated and analyzed by immunoblotting using (A) anti-Bax or (B) anti-cytochrome c as primary antibodies and HRP-conjugated anti-isotype secondary antibody. Blots were also probed for COX IV as a control marker for the mitochondrial fractions and GAPDH as a control marker for the cytosolic fractions. The blots shown are representative of three independent experiments. Asterisks indicate a clear increase induced by EPEC.

    Article Snippet: The human epithelial HEp-2 (ATCC CCL23) or HT-29 (ATCC HTB-38) cell lines were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT), 1% nonessential amino acids, 5 mM l -glutamine, penicillin (100 U/ml), and streptomycin (100 µg/ml).

    Techniques: Translocation Assay, Infection, Marker

    EspC preferentially induces caspase-9 activity. (A) Activity of caspase-8 and (B) caspase-9 induced by EspC. HEp-2 cells were infected with the EPEC WT, Δ espC , Δ espC /p espC , or Δ escN strain at an MOI of 10 for 4 h. The whole-cell lysates were subjected to caspase activity assay by using specific substrates for each caspase as described in Materials and Methods. Activity is represented as fold change relative to uninfected cells. Data are expressed as the mean ± SEM from at least 3 independent experiments. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparison test for comparison to the WT strain (*, P

    Journal: mBio

    Article Title: EspC, an Autotransporter Protein Secreted by Enteropathogenic Escherichia coli, Causes Apoptosis and Necrosis through Caspase and Calpain Activation, Including Direct Procaspase-3 Cleavage

    doi: 10.1128/mBio.00479-16

    Figure Lengend Snippet: EspC preferentially induces caspase-9 activity. (A) Activity of caspase-8 and (B) caspase-9 induced by EspC. HEp-2 cells were infected with the EPEC WT, Δ espC , Δ espC /p espC , or Δ escN strain at an MOI of 10 for 4 h. The whole-cell lysates were subjected to caspase activity assay by using specific substrates for each caspase as described in Materials and Methods. Activity is represented as fold change relative to uninfected cells. Data are expressed as the mean ± SEM from at least 3 independent experiments. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparison test for comparison to the WT strain (*, P

    Article Snippet: The human epithelial HEp-2 (ATCC CCL23) or HT-29 (ATCC HTB-38) cell lines were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT), 1% nonessential amino acids, 5 mM l -glutamine, penicillin (100 U/ml), and streptomycin (100 µg/ml).

    Techniques: Activity Assay, Infection, Caspase Activity Assay

    EspC induces PARP proteolysis and DNA nuclear fragmentation. (A) EspC induces PARP cleavage. HEp-2 cells were infected with the bacterial strains as indicated at an MOI of 10 and for the indicated times. Infected cells were lysed, and proteins were analyzed by immunoblotting using anti-cleaved PARP and anti-β-actin as primary antibodies and HRP-conjugated anti-isotype secondary antibody. (B) EspC induces DNA nuclear fragmentation. HEp-2 cells were infected with the EPEC WT, Δ espC , Δ espC /p espC , Δ espC /p espC S256I , or Δ escN strain at an MOI of 10 for 4 h. The cell cycle was measured by using propidium iodide (PI) staining; results are presented as histograms. (C) EspC induces an increase in the sub-G 1 cell population. Cell distribution was measured by fluorescence intensity in the sub-G 1 phase. Results are shown as percentages of the sub-G 1 population. Data are expressed as the mean ± SEM from at least 3 independent experiments. Statistical analyses were performed using unpaired t test (*, P

    Journal: mBio

    Article Title: EspC, an Autotransporter Protein Secreted by Enteropathogenic Escherichia coli, Causes Apoptosis and Necrosis through Caspase and Calpain Activation, Including Direct Procaspase-3 Cleavage

    doi: 10.1128/mBio.00479-16

    Figure Lengend Snippet: EspC induces PARP proteolysis and DNA nuclear fragmentation. (A) EspC induces PARP cleavage. HEp-2 cells were infected with the bacterial strains as indicated at an MOI of 10 and for the indicated times. Infected cells were lysed, and proteins were analyzed by immunoblotting using anti-cleaved PARP and anti-β-actin as primary antibodies and HRP-conjugated anti-isotype secondary antibody. (B) EspC induces DNA nuclear fragmentation. HEp-2 cells were infected with the EPEC WT, Δ espC , Δ espC /p espC , Δ espC /p espC S256I , or Δ escN strain at an MOI of 10 for 4 h. The cell cycle was measured by using propidium iodide (PI) staining; results are presented as histograms. (C) EspC induces an increase in the sub-G 1 cell population. Cell distribution was measured by fluorescence intensity in the sub-G 1 phase. Results are shown as percentages of the sub-G 1 population. Data are expressed as the mean ± SEM from at least 3 independent experiments. Statistical analyses were performed using unpaired t test (*, P

    Article Snippet: The human epithelial HEp-2 (ATCC CCL23) or HT-29 (ATCC HTB-38) cell lines were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT), 1% nonessential amino acids, 5 mM l -glutamine, penicillin (100 U/ml), and streptomycin (100 µg/ml).

    Techniques: Infection, Staining, Fluorescence

    EspC induces a decrease in Bcl-2 protein levels. (A) Kinetics of detection of Bcl-2 in cells infected with the EPEC WT and Δ espC mutant. HEp-2 cells were infected with bacterial strains as indicated and for the indicated lengths of time. The asterisk indicates a clear Bcl-2 decrease induced by EPEC (B) detection of Bcl-2 at 4 h of infection with the EPEC WT, Δ espC , Δ espC /p espC , Δ espC /p espC S256I , or Δ escN strain at an MOI of 10. Infected cells were lysed, and proteins were analyzed by immunoblotting using anti-Bcl-2 and anti-β-actin as primary antibodies and HRP-conjugated anti-isotype secondary antibody. Densitometries of three immunoblots were plotted. Results are expressed as the mean ± SEM from at least 3 independent experiments. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparison test for comparison with mock-infected cells (**, P

    Journal: mBio

    Article Title: EspC, an Autotransporter Protein Secreted by Enteropathogenic Escherichia coli, Causes Apoptosis and Necrosis through Caspase and Calpain Activation, Including Direct Procaspase-3 Cleavage

    doi: 10.1128/mBio.00479-16

    Figure Lengend Snippet: EspC induces a decrease in Bcl-2 protein levels. (A) Kinetics of detection of Bcl-2 in cells infected with the EPEC WT and Δ espC mutant. HEp-2 cells were infected with bacterial strains as indicated and for the indicated lengths of time. The asterisk indicates a clear Bcl-2 decrease induced by EPEC (B) detection of Bcl-2 at 4 h of infection with the EPEC WT, Δ espC , Δ espC /p espC , Δ espC /p espC S256I , or Δ escN strain at an MOI of 10. Infected cells were lysed, and proteins were analyzed by immunoblotting using anti-Bcl-2 and anti-β-actin as primary antibodies and HRP-conjugated anti-isotype secondary antibody. Densitometries of three immunoblots were plotted. Results are expressed as the mean ± SEM from at least 3 independent experiments. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparison test for comparison with mock-infected cells (**, P

    Article Snippet: The human epithelial HEp-2 (ATCC CCL23) or HT-29 (ATCC HTB-38) cell lines were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT), 1% nonessential amino acids, 5 mM l -glutamine, penicillin (100 U/ml), and streptomycin (100 µg/ml).

    Techniques: Infection, Mutagenesis, Western Blot

    EspC produces cell death on epithelial cells. (A to F) Cytotoxic effects induced by EspC-producing EPEC. Untreated HEp-2 cells were used as the control (A), and HEp-2 cells were infected with the EPEC wild-type (WT) strain (E2348/69) (B), Δ espC mutant (C), Δ espC /p espC complemented strain (D), Δ espC /p espC S256I complemented strain (E), or Δ escN mutant (F) for 4 h. Infected cells were fixed and permeabilized. Cells were immunostained with anti-EspC antibody, followed by a secondary antibody conjugated to fluorescein isothiocyanate (FITC), and the actin cytoskeleton was detected with rhodamine-phalloidin. Slides were observed using a Leica TCS SP8 confocal microscope. Scale bar, 20 µm. (G) Cell death induced by EspC-producing EPEC. HEp-2 cells were infected with the EPEC WT, Δ espC , Δ espC /p espC , Δ espC /p espC S256I , or Δ escN strain at an MOI of 10 for different lengths of time. Cells were harvested and stained with propidium iodide (PI) to perform a PI exclusion assay by flow cytometry. Data are expressed as the mean ± SEM from at least 3 independent experiments. Statistical analysis was performed using two-way ANOVA followed by Dunnett’s multiple comparison test for comparison to the WT strain (*, P

    Journal: mBio

    Article Title: EspC, an Autotransporter Protein Secreted by Enteropathogenic Escherichia coli, Causes Apoptosis and Necrosis through Caspase and Calpain Activation, Including Direct Procaspase-3 Cleavage

    doi: 10.1128/mBio.00479-16

    Figure Lengend Snippet: EspC produces cell death on epithelial cells. (A to F) Cytotoxic effects induced by EspC-producing EPEC. Untreated HEp-2 cells were used as the control (A), and HEp-2 cells were infected with the EPEC wild-type (WT) strain (E2348/69) (B), Δ espC mutant (C), Δ espC /p espC complemented strain (D), Δ espC /p espC S256I complemented strain (E), or Δ escN mutant (F) for 4 h. Infected cells were fixed and permeabilized. Cells were immunostained with anti-EspC antibody, followed by a secondary antibody conjugated to fluorescein isothiocyanate (FITC), and the actin cytoskeleton was detected with rhodamine-phalloidin. Slides were observed using a Leica TCS SP8 confocal microscope. Scale bar, 20 µm. (G) Cell death induced by EspC-producing EPEC. HEp-2 cells were infected with the EPEC WT, Δ espC , Δ espC /p espC , Δ espC /p espC S256I , or Δ escN strain at an MOI of 10 for different lengths of time. Cells were harvested and stained with propidium iodide (PI) to perform a PI exclusion assay by flow cytometry. Data are expressed as the mean ± SEM from at least 3 independent experiments. Statistical analysis was performed using two-way ANOVA followed by Dunnett’s multiple comparison test for comparison to the WT strain (*, P

    Article Snippet: The human epithelial HEp-2 (ATCC CCL23) or HT-29 (ATCC HTB-38) cell lines were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT), 1% nonessential amino acids, 5 mM l -glutamine, penicillin (100 U/ml), and streptomycin (100 µg/ml).

    Techniques: Infection, Mutagenesis, Microscopy, Staining, Exclusion Assay, Flow Cytometry, Cytometry

    EspC induces calpain activity during EPEC infection. (A) Measurement of calpain activity induced by EspC. HEp-2 cells were pretreated with 20 µM fluorescent substrate t -BOC-Leu-Met-CMAC upon cleavage by calpain. Cells were infected with the EPEC WT (E2348/69) or Δ espC mutant at an MOI of 10 for 4 h. Calpain activity was measured as increase in fluorescence intensity. (B) Kinetics of calpain activity induced by the EPEC WT or Δ espC mutant. Cells were infected with the bacterial strains at an MOI of 10 for the indicated times. Calpain activity was measured as increase of mean fluorescence intensity. All measurements were expressed relative to the calpain activity measured in mock-infected cells. Activity is represented as fold change relative to uninfected cells. (C) EspC causes an increase in calpain activity. HEp-2 cells were infected with the different bacterial strains as indicated at an MOI of 10 for 3 h. MDL28170, a specific calpain inhibitor, was included to demonstrate that the increase of fluorescence is specific to calpain-like proteases. Data are shown as the mean ± SEM from at least 3 independent experiments. Statistical analyses were performed using (B) unpaired t test (*, P

    Journal: mBio

    Article Title: EspC, an Autotransporter Protein Secreted by Enteropathogenic Escherichia coli, Causes Apoptosis and Necrosis through Caspase and Calpain Activation, Including Direct Procaspase-3 Cleavage

    doi: 10.1128/mBio.00479-16

    Figure Lengend Snippet: EspC induces calpain activity during EPEC infection. (A) Measurement of calpain activity induced by EspC. HEp-2 cells were pretreated with 20 µM fluorescent substrate t -BOC-Leu-Met-CMAC upon cleavage by calpain. Cells were infected with the EPEC WT (E2348/69) or Δ espC mutant at an MOI of 10 for 4 h. Calpain activity was measured as increase in fluorescence intensity. (B) Kinetics of calpain activity induced by the EPEC WT or Δ espC mutant. Cells were infected with the bacterial strains at an MOI of 10 for the indicated times. Calpain activity was measured as increase of mean fluorescence intensity. All measurements were expressed relative to the calpain activity measured in mock-infected cells. Activity is represented as fold change relative to uninfected cells. (C) EspC causes an increase in calpain activity. HEp-2 cells were infected with the different bacterial strains as indicated at an MOI of 10 for 3 h. MDL28170, a specific calpain inhibitor, was included to demonstrate that the increase of fluorescence is specific to calpain-like proteases. Data are shown as the mean ± SEM from at least 3 independent experiments. Statistical analyses were performed using (B) unpaired t test (*, P

    Article Snippet: The human epithelial HEp-2 (ATCC CCL23) or HT-29 (ATCC HTB-38) cell lines were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT), 1% nonessential amino acids, 5 mM l -glutamine, penicillin (100 U/ml), and streptomycin (100 µg/ml).

    Techniques: Activity Assay, Infection, Mutagenesis, Fluorescence

    EspC induces an increase in intracellular Ca 2+ associated with necrosis. (A) Intracellular calcium chelation causes a decrease in the necrosis induced by EspC. HEp-2 cells were pretreated with 20 µM BAPTA-AM or only DMSO for 1 h. HEp-2 cells were infected with the indicated bacterial strains at an MOI of 10 for 4 h. Cells were harvested and labeled with annexin V and PI and analyzed by flow cytometry. Annexin V-negative and PI-positive cells were considered necrotic cells. Data are shown as the mean ± SEM from at least 3 independent experiments. Statistical analysis was performed using two-way ANOVA followed by Bonferroni’s multiple comparison test (*, P

    Journal: mBio

    Article Title: EspC, an Autotransporter Protein Secreted by Enteropathogenic Escherichia coli, Causes Apoptosis and Necrosis through Caspase and Calpain Activation, Including Direct Procaspase-3 Cleavage

    doi: 10.1128/mBio.00479-16

    Figure Lengend Snippet: EspC induces an increase in intracellular Ca 2+ associated with necrosis. (A) Intracellular calcium chelation causes a decrease in the necrosis induced by EspC. HEp-2 cells were pretreated with 20 µM BAPTA-AM or only DMSO for 1 h. HEp-2 cells were infected with the indicated bacterial strains at an MOI of 10 for 4 h. Cells were harvested and labeled with annexin V and PI and analyzed by flow cytometry. Annexin V-negative and PI-positive cells were considered necrotic cells. Data are shown as the mean ± SEM from at least 3 independent experiments. Statistical analysis was performed using two-way ANOVA followed by Bonferroni’s multiple comparison test (*, P

    Article Snippet: The human epithelial HEp-2 (ATCC CCL23) or HT-29 (ATCC HTB-38) cell lines were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT), 1% nonessential amino acids, 5 mM l -glutamine, penicillin (100 U/ml), and streptomycin (100 µg/ml).

    Techniques: Infection, Labeling, Flow Cytometry, Cytometry

    Overexpressed hsa-miR-138-2-3p activated JNK1/MAPK pathway. The vertical and horizontal axis stand for Gary value and treaments, respectively. It suggested that the expression of JNK1 between 100nM-TR and 100nMN-CR of Hep-2 (A), M2e (B) and TU212 (C) CSCs were significant difference (** P

    Journal: PeerJ

    Article Title: Radiosensitization effect of hsa-miR-138-2-3p on human laryngeal cancer stem cells

    doi: 10.7717/peerj.3233

    Figure Lengend Snippet: Overexpressed hsa-miR-138-2-3p activated JNK1/MAPK pathway. The vertical and horizontal axis stand for Gary value and treaments, respectively. It suggested that the expression of JNK1 between 100nM-TR and 100nMN-CR of Hep-2 (A), M2e (B) and TU212 (C) CSCs were significant difference (** P

    Article Snippet: Laryngeal cancer sphere culture Three human laryngeal squamous cancer cell lines, Hep-2, TU212 and M2e, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

    Techniques: Expressing

    Overexpressed hsa-miR-138-2-3p induced cell apoptosis after radiation by flow cytometry. (A) and (C) show the cell apoptosis analysis of 100nM-TR and 100nMN-CR of Hep-2 cell line after radiation, respectively; (B) and (E) show the cell apoptosis analysis of 100nM-TR and 100nMN-CR of M2e cell line after radiation, respectively; (C) and (F) show the cell apoptosis analysis of 100nM-TR and 100nMN-CR of TU212 cell line after radiation, respectively. The vertical and horizontal axis stand for PI positive area and FITC positive area, respectively. Identified by flow cytometry, cells were divided into four sections: Q1: Annexin V-FITC-PI+, was representative of mechanical error; Q2: Annexin V-FITC+ PI+, was representative of late apoptosis or necrosis cells; Q3: Annexin V-FITC- PI-, was representative of living cells; Q4: Annexin V-FITC+ PI-, was representative of early apoptosis cells. (A–C) were shown the proportion of early apoptosis (29.6%, 33.6%, 23.5%) and late apoptosis (25.2%, 17.7%, 18.1%) of Hep-2, M2e, and TU212 CSCs induced by transfection of 100nM hsa-miR-138-2-3p were larger than that of induction by transfection of 100nM nonsense oligonucleotides ((D) early apoptosis of Hep-2 CSCs was 10.8%; (E) early apoptosis of M2e CSCs was 8.0%; (F) early apoptosis of TU212 CSCs was 3.7%; (D) late apoptosis of Hep-2 CSCs was 8.0%; (E) late apoptosis of M2e CSCs was 6.7%; (F) late apoptosis of TU212 CSCs was 5.8%), respectively after radiation.

    Journal: PeerJ

    Article Title: Radiosensitization effect of hsa-miR-138-2-3p on human laryngeal cancer stem cells

    doi: 10.7717/peerj.3233

    Figure Lengend Snippet: Overexpressed hsa-miR-138-2-3p induced cell apoptosis after radiation by flow cytometry. (A) and (C) show the cell apoptosis analysis of 100nM-TR and 100nMN-CR of Hep-2 cell line after radiation, respectively; (B) and (E) show the cell apoptosis analysis of 100nM-TR and 100nMN-CR of M2e cell line after radiation, respectively; (C) and (F) show the cell apoptosis analysis of 100nM-TR and 100nMN-CR of TU212 cell line after radiation, respectively. The vertical and horizontal axis stand for PI positive area and FITC positive area, respectively. Identified by flow cytometry, cells were divided into four sections: Q1: Annexin V-FITC-PI+, was representative of mechanical error; Q2: Annexin V-FITC+ PI+, was representative of late apoptosis or necrosis cells; Q3: Annexin V-FITC- PI-, was representative of living cells; Q4: Annexin V-FITC+ PI-, was representative of early apoptosis cells. (A–C) were shown the proportion of early apoptosis (29.6%, 33.6%, 23.5%) and late apoptosis (25.2%, 17.7%, 18.1%) of Hep-2, M2e, and TU212 CSCs induced by transfection of 100nM hsa-miR-138-2-3p were larger than that of induction by transfection of 100nM nonsense oligonucleotides ((D) early apoptosis of Hep-2 CSCs was 10.8%; (E) early apoptosis of M2e CSCs was 8.0%; (F) early apoptosis of TU212 CSCs was 3.7%; (D) late apoptosis of Hep-2 CSCs was 8.0%; (E) late apoptosis of M2e CSCs was 6.7%; (F) late apoptosis of TU212 CSCs was 5.8%), respectively after radiation.

    Article Snippet: Laryngeal cancer sphere culture Three human laryngeal squamous cancer cell lines, Hep-2, TU212 and M2e, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

    Techniques: Flow Cytometry, Cytometry, Transfection

    Overexpressed hsa-miR-138-2-3p reduced survival fraction after radiation. (A–C) shows the comparison of survival analysis among 100nM-TR, 100nMN-CR and non-transfection of Hep-2, M2e, and TU212 cell lines after radiation, respectively. The vertical and horizontal axis stand for survival fraction and does(Gy), respectively. 100nM-TR, 100nM-CR, and non-transfection of Hep-2, M2e and TU212 were treated with 0, 2, 4, 6, 8 Gy X-ray irradiation, respectively, and the survival fraction of 100nM-TR of all laryngeal CSCs were lower than that of 100nMN-CR and non-transfection, and the difference between 100nM-TR and non-transfection were statistically significant (* P

    Journal: PeerJ

    Article Title: Radiosensitization effect of hsa-miR-138-2-3p on human laryngeal cancer stem cells

    doi: 10.7717/peerj.3233

    Figure Lengend Snippet: Overexpressed hsa-miR-138-2-3p reduced survival fraction after radiation. (A–C) shows the comparison of survival analysis among 100nM-TR, 100nMN-CR and non-transfection of Hep-2, M2e, and TU212 cell lines after radiation, respectively. The vertical and horizontal axis stand for survival fraction and does(Gy), respectively. 100nM-TR, 100nM-CR, and non-transfection of Hep-2, M2e and TU212 were treated with 0, 2, 4, 6, 8 Gy X-ray irradiation, respectively, and the survival fraction of 100nM-TR of all laryngeal CSCs were lower than that of 100nMN-CR and non-transfection, and the difference between 100nM-TR and non-transfection were statistically significant (* P

    Article Snippet: Laryngeal cancer sphere culture Three human laryngeal squamous cancer cell lines, Hep-2, TU212 and M2e, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

    Techniques: Transfection, Irradiation

    Cell transfection efficiency evaluated by Flow Cytometry. (A–C) show the transfection efficiency of FAM-CR teams of Hep-2, M2e, and TU212 cell line, respectively, and the percentages of FAM-positive cells (represented by “P2”) were identified by flow cytometry. The transfection efficiency of Hep-2, M2e, and TU212 cell lines were 82.9%, 91.5% and 90.5%, respectively, and it was indicated that hsa-miR-138-2-3p and nonsense oligonucleotides were successfully transfected into the laryngeal CSCs with high efficiency.

    Journal: PeerJ

    Article Title: Radiosensitization effect of hsa-miR-138-2-3p on human laryngeal cancer stem cells

    doi: 10.7717/peerj.3233

    Figure Lengend Snippet: Cell transfection efficiency evaluated by Flow Cytometry. (A–C) show the transfection efficiency of FAM-CR teams of Hep-2, M2e, and TU212 cell line, respectively, and the percentages of FAM-positive cells (represented by “P2”) were identified by flow cytometry. The transfection efficiency of Hep-2, M2e, and TU212 cell lines were 82.9%, 91.5% and 90.5%, respectively, and it was indicated that hsa-miR-138-2-3p and nonsense oligonucleotides were successfully transfected into the laryngeal CSCs with high efficiency.

    Article Snippet: Laryngeal cancer sphere culture Three human laryngeal squamous cancer cell lines, Hep-2, TU212 and M2e, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

    Techniques: Transfection, Flow Cytometry, Cytometry

    Overexpressed hsa-miR-138-2-3p arrested cell cycle at G1/S phase after radiation by flow cytometry. (A) and (D) show the cell cycle analysis of 100nM-TR and 100nMN-CR of Hep-2 cell line after radiation, respectively; (B) and (E) show the cell cycle analysis of 100nM-TR and 100nMN-CR of M2e cell line after radiation, respectively; (C) and (F) show the cell cycle analysis of 100nM-TR and 100nMN-CR of TU212 cell line after radiation, respectively. The vertical and horizontal axis stand for cell numbers and cell division cycle, respectively. The percentage of Hep-2, M2e, and TU212 CSCs induced by transfection of 100nM hsa-miR-138-2-3p in G1 phase (68.68%, 65.95%, 65.24%) were more than that induced by transfection of 100nM nonsense oligonucleotides (55.44%, 56.90%, 59.23%), respectively after radiation. While, the percentage of Hep-2, M2e, and TU212 CSCs induced by transfection of 100nM hsa-miR-138-2-3p in S phase (23.32%, 26.05%, 26.76%) were less than that induced by transfection of 100nM nonsense oligonucleotides (36.56%, 37.25%, 37.96%), respectively, after radiation. The percentage of M2e, and TU212 CSCs induced by transfection of 100nM hsa-miR-138-2-3p in G2 phase (8%, 8%) were higher than that induced by transfection of 100nM nonsense oligonucleotides (5.85%, 2.81%), respectively, after radiation, but the percentage of Hep-2 CSCs induced by transfection of 100nM hsa-miR-138-2-3p in G2 phase (8%) was the same as that induced by transfection of 100nM nonsense oligonucleotides (8%) after radiation.

    Journal: PeerJ

    Article Title: Radiosensitization effect of hsa-miR-138-2-3p on human laryngeal cancer stem cells

    doi: 10.7717/peerj.3233

    Figure Lengend Snippet: Overexpressed hsa-miR-138-2-3p arrested cell cycle at G1/S phase after radiation by flow cytometry. (A) and (D) show the cell cycle analysis of 100nM-TR and 100nMN-CR of Hep-2 cell line after radiation, respectively; (B) and (E) show the cell cycle analysis of 100nM-TR and 100nMN-CR of M2e cell line after radiation, respectively; (C) and (F) show the cell cycle analysis of 100nM-TR and 100nMN-CR of TU212 cell line after radiation, respectively. The vertical and horizontal axis stand for cell numbers and cell division cycle, respectively. The percentage of Hep-2, M2e, and TU212 CSCs induced by transfection of 100nM hsa-miR-138-2-3p in G1 phase (68.68%, 65.95%, 65.24%) were more than that induced by transfection of 100nM nonsense oligonucleotides (55.44%, 56.90%, 59.23%), respectively after radiation. While, the percentage of Hep-2, M2e, and TU212 CSCs induced by transfection of 100nM hsa-miR-138-2-3p in S phase (23.32%, 26.05%, 26.76%) were less than that induced by transfection of 100nM nonsense oligonucleotides (36.56%, 37.25%, 37.96%), respectively, after radiation. The percentage of M2e, and TU212 CSCs induced by transfection of 100nM hsa-miR-138-2-3p in G2 phase (8%, 8%) were higher than that induced by transfection of 100nM nonsense oligonucleotides (5.85%, 2.81%), respectively, after radiation, but the percentage of Hep-2 CSCs induced by transfection of 100nM hsa-miR-138-2-3p in G2 phase (8%) was the same as that induced by transfection of 100nM nonsense oligonucleotides (8%) after radiation.

    Article Snippet: Laryngeal cancer sphere culture Three human laryngeal squamous cancer cell lines, Hep-2, TU212 and M2e, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

    Techniques: Flow Cytometry, Cytometry, Cell Cycle Assay, Transfection

    Overexpressed hsa-miR-138-2-3p promoted DNA damage after radiation by Comet assay. (A) and (D) show the DNA damage analysis of 100nM-TR and 100nMN-CR of Hep-2 cell line after radiation, respectively; (B) and (E) show the DNA damage analysis of 100nM-TR and 100nMN-CR of M2e cell line after radiation, respectively; (C) and (F) show the DNA damage analysis of 100nM-TR and 100nMN-CR of TU212 cell line after radiation, respectively. The appearance of “comet” with fragmented DNA (tail) being separated from undamaged nuclear DNA (head) was seen in 100nM-TR and 100nMN-CR of Hep-2, M2e, and TU212 CSCs after radiation. It was found that the “heads” of “comet” of 100nM-TR were smaller than that of 100nMN-CR, while the “tails” of “comet” of 100nM-TR were longer than that of 100nMN-CR. These data were indicated that the DNA damage of 100nM-TR were more serious than that of 100nM-CR in laryngeal CSCs after radiation.

    Journal: PeerJ

    Article Title: Radiosensitization effect of hsa-miR-138-2-3p on human laryngeal cancer stem cells

    doi: 10.7717/peerj.3233

    Figure Lengend Snippet: Overexpressed hsa-miR-138-2-3p promoted DNA damage after radiation by Comet assay. (A) and (D) show the DNA damage analysis of 100nM-TR and 100nMN-CR of Hep-2 cell line after radiation, respectively; (B) and (E) show the DNA damage analysis of 100nM-TR and 100nMN-CR of M2e cell line after radiation, respectively; (C) and (F) show the DNA damage analysis of 100nM-TR and 100nMN-CR of TU212 cell line after radiation, respectively. The appearance of “comet” with fragmented DNA (tail) being separated from undamaged nuclear DNA (head) was seen in 100nM-TR and 100nMN-CR of Hep-2, M2e, and TU212 CSCs after radiation. It was found that the “heads” of “comet” of 100nM-TR were smaller than that of 100nMN-CR, while the “tails” of “comet” of 100nM-TR were longer than that of 100nMN-CR. These data were indicated that the DNA damage of 100nM-TR were more serious than that of 100nM-CR in laryngeal CSCs after radiation.

    Article Snippet: Laryngeal cancer sphere culture Three human laryngeal squamous cancer cell lines, Hep-2, TU212 and M2e, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

    Techniques: Single Cell Gel Electrophoresis

    Overexpressed hsa-miR-138-2-3p inhibited Wnt/ β -catenin pathway. The vertical and horizontal axis stand for Gary value and treaments, respectively. It suggested that the expression of β -catenin between 100nM-TR and 100nMN-CR of Hep-2 (A), M2e (B) and TU212 (C) CSCs were significant difference (*** P

    Journal: PeerJ

    Article Title: Radiosensitization effect of hsa-miR-138-2-3p on human laryngeal cancer stem cells

    doi: 10.7717/peerj.3233

    Figure Lengend Snippet: Overexpressed hsa-miR-138-2-3p inhibited Wnt/ β -catenin pathway. The vertical and horizontal axis stand for Gary value and treaments, respectively. It suggested that the expression of β -catenin between 100nM-TR and 100nMN-CR of Hep-2 (A), M2e (B) and TU212 (C) CSCs were significant difference (*** P

    Article Snippet: Laryngeal cancer sphere culture Three human laryngeal squamous cancer cell lines, Hep-2, TU212 and M2e, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

    Techniques: Expressing

    Overexpressed hsa-miR-138-2-3p down-regulated expression of β -catenin. The expression of β -catenin in 100nM-TR of Hep-2 (A), M2e (B) and TU212 (C) CSCs were reduced more than that in 100nMN-CR.

    Journal: PeerJ

    Article Title: Radiosensitization effect of hsa-miR-138-2-3p on human laryngeal cancer stem cells

    doi: 10.7717/peerj.3233

    Figure Lengend Snippet: Overexpressed hsa-miR-138-2-3p down-regulated expression of β -catenin. The expression of β -catenin in 100nM-TR of Hep-2 (A), M2e (B) and TU212 (C) CSCs were reduced more than that in 100nMN-CR.

    Article Snippet: Laryngeal cancer sphere culture Three human laryngeal squamous cancer cell lines, Hep-2, TU212 and M2e, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

    Techniques: Expressing

    Overexpressed hsa-miR-138-2-3p up-regulated expression of JNK1 and p38. The expression of JNK1 and p38 in 100nM-TR of Hep-2 (A), M2e (B) and TU212 (C) CSCs were improved more than that in 100nMN-CR.

    Journal: PeerJ

    Article Title: Radiosensitization effect of hsa-miR-138-2-3p on human laryngeal cancer stem cells

    doi: 10.7717/peerj.3233

    Figure Lengend Snippet: Overexpressed hsa-miR-138-2-3p up-regulated expression of JNK1 and p38. The expression of JNK1 and p38 in 100nM-TR of Hep-2 (A), M2e (B) and TU212 (C) CSCs were improved more than that in 100nMN-CR.

    Article Snippet: Laryngeal cancer sphere culture Three human laryngeal squamous cancer cell lines, Hep-2, TU212 and M2e, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

    Techniques: Expressing

    Overexpressed hsa-miR-138-2-3p arrested cell cycle at G1/S phase after radiation. (A) and (B) show the cell cycle analysis of 100nM-TR and 100nMN-CR of Hep-2 cell line after radiation, respectively; (C) and (D) show the cell cycle analysis of 100nM-TR and 100nMN-CR of M2e cell line after radiation, respectively; (E) and (F) show the cell cycle analysis of 100nM-TR and 100nMN-CR of TU212 cell line after radiation, respectively. The vertical and horizontal axis stand for proportions and cell division phases, respectively. The proportion of Hep-2, M2e, and TU212 CSCs induced by transfection of 100nM hsa-miR-138-2-3p in G1 phase were higher than that induced by transfection of 100nM nonsense oligonucleotides, respectively after radiation. While, the percentage of Hep-2, M2e, and TU212 CSCs induced by transfection of 100nM hsa-miR-138-2-3p in S phase were less than that induced by transfection of 100nM nonsense oligonucleotides, respectively after radiation. The percentage of M2e, and TU212 CSCs induced by transfection of 100nM hsa-miR-138-2-3p in G2 phase were more than that induced by transfection of 100nM nonsense oligonucleotides, respectively after radiation, but the percentage of Hep-2 CSCs induced by transfection of 100nM hsa-miR-138-2-3p in G2 phase was the same as that induced by transfection of 100nM nonsense oligonucleotides after radiation. However, all differences between them were not statistically significant ( P > 0.05).

    Journal: PeerJ

    Article Title: Radiosensitization effect of hsa-miR-138-2-3p on human laryngeal cancer stem cells

    doi: 10.7717/peerj.3233

    Figure Lengend Snippet: Overexpressed hsa-miR-138-2-3p arrested cell cycle at G1/S phase after radiation. (A) and (B) show the cell cycle analysis of 100nM-TR and 100nMN-CR of Hep-2 cell line after radiation, respectively; (C) and (D) show the cell cycle analysis of 100nM-TR and 100nMN-CR of M2e cell line after radiation, respectively; (E) and (F) show the cell cycle analysis of 100nM-TR and 100nMN-CR of TU212 cell line after radiation, respectively. The vertical and horizontal axis stand for proportions and cell division phases, respectively. The proportion of Hep-2, M2e, and TU212 CSCs induced by transfection of 100nM hsa-miR-138-2-3p in G1 phase were higher than that induced by transfection of 100nM nonsense oligonucleotides, respectively after radiation. While, the percentage of Hep-2, M2e, and TU212 CSCs induced by transfection of 100nM hsa-miR-138-2-3p in S phase were less than that induced by transfection of 100nM nonsense oligonucleotides, respectively after radiation. The percentage of M2e, and TU212 CSCs induced by transfection of 100nM hsa-miR-138-2-3p in G2 phase were more than that induced by transfection of 100nM nonsense oligonucleotides, respectively after radiation, but the percentage of Hep-2 CSCs induced by transfection of 100nM hsa-miR-138-2-3p in G2 phase was the same as that induced by transfection of 100nM nonsense oligonucleotides after radiation. However, all differences between them were not statistically significant ( P > 0.05).

    Article Snippet: Laryngeal cancer sphere culture Three human laryngeal squamous cancer cell lines, Hep-2, TU212 and M2e, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

    Techniques: Cell Cycle Assay, Transfection

    Overexpressed hsa-miR-138-2-3p promoted DNA damage after radiation. (A) shows the comparison of DNA damage between 100nM-TR and 100nMN-CR of Hep-2 cell line after radiation, respectively; (B) shows the comparison of DNA damage between 100nM-TR and 100nMN-CR of M2e cell line after radiation, respectively; (C) shows the comparison of DNA damage between 100nM-TR and 100nMN-CR of TU212 cell line after radiation, respectively. The vertical and horizontal axis stand for Tail movement (%) and treaments, respectively. The tail movement of 100nM-TR of all laryngeal cell lines were higher than that of 100nMN-CR, and the differences between 100nM-TR and 100nMN-CR of Hep-2 CSCs were statistically significant (** P

    Journal: PeerJ

    Article Title: Radiosensitization effect of hsa-miR-138-2-3p on human laryngeal cancer stem cells

    doi: 10.7717/peerj.3233

    Figure Lengend Snippet: Overexpressed hsa-miR-138-2-3p promoted DNA damage after radiation. (A) shows the comparison of DNA damage between 100nM-TR and 100nMN-CR of Hep-2 cell line after radiation, respectively; (B) shows the comparison of DNA damage between 100nM-TR and 100nMN-CR of M2e cell line after radiation, respectively; (C) shows the comparison of DNA damage between 100nM-TR and 100nMN-CR of TU212 cell line after radiation, respectively. The vertical and horizontal axis stand for Tail movement (%) and treaments, respectively. The tail movement of 100nM-TR of all laryngeal cell lines were higher than that of 100nMN-CR, and the differences between 100nM-TR and 100nMN-CR of Hep-2 CSCs were statistically significant (** P

    Article Snippet: Laryngeal cancer sphere culture Three human laryngeal squamous cancer cell lines, Hep-2, TU212 and M2e, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

    Techniques:

    Overexpressed hsa-miR-138-2-3p inhibited Hippo/YAP1 pathway. The vertical and horizontal axis stand for Gary value and treaments, respectively. It suggested that the expression of YAP1 between 100nM-TR and 100nMN-CR of Hep-2 (A), M2e (B) and TU212 (C) CSCs were significant difference (*** P

    Journal: PeerJ

    Article Title: Radiosensitization effect of hsa-miR-138-2-3p on human laryngeal cancer stem cells

    doi: 10.7717/peerj.3233

    Figure Lengend Snippet: Overexpressed hsa-miR-138-2-3p inhibited Hippo/YAP1 pathway. The vertical and horizontal axis stand for Gary value and treaments, respectively. It suggested that the expression of YAP1 between 100nM-TR and 100nMN-CR of Hep-2 (A), M2e (B) and TU212 (C) CSCs were significant difference (*** P

    Article Snippet: Laryngeal cancer sphere culture Three human laryngeal squamous cancer cell lines, Hep-2, TU212 and M2e, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

    Techniques: Expressing

    Overexpressed hsa-miR-138-2-3p activated p38/MAPK pathway. The vertical and horizontal axis stand for Gary value and treaments, respectively. It suggests that the expression of p38 between 100nM-TR and 100nMN-CR of Hep-2 (A), M2e (B) and TU212 (C) CSCs were significantly difference (* P

    Journal: PeerJ

    Article Title: Radiosensitization effect of hsa-miR-138-2-3p on human laryngeal cancer stem cells

    doi: 10.7717/peerj.3233

    Figure Lengend Snippet: Overexpressed hsa-miR-138-2-3p activated p38/MAPK pathway. The vertical and horizontal axis stand for Gary value and treaments, respectively. It suggests that the expression of p38 between 100nM-TR and 100nMN-CR of Hep-2 (A), M2e (B) and TU212 (C) CSCs were significantly difference (* P

    Article Snippet: Laryngeal cancer sphere culture Three human laryngeal squamous cancer cell lines, Hep-2, TU212 and M2e, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

    Techniques: Expressing

    Overexpressed hsa-miR-138-2-3p down-regulated expression of YAP1. The expression of YAP1 in 100nM-TR of Hep-2 (A), M2e (B) and TU212 (C) CSCs were reduced more than that in 100nMN-CR.

    Journal: PeerJ

    Article Title: Radiosensitization effect of hsa-miR-138-2-3p on human laryngeal cancer stem cells

    doi: 10.7717/peerj.3233

    Figure Lengend Snippet: Overexpressed hsa-miR-138-2-3p down-regulated expression of YAP1. The expression of YAP1 in 100nM-TR of Hep-2 (A), M2e (B) and TU212 (C) CSCs were reduced more than that in 100nMN-CR.

    Article Snippet: Laryngeal cancer sphere culture Three human laryngeal squamous cancer cell lines, Hep-2, TU212 and M2e, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

    Techniques: Expressing

    Overexpressed hsa-miR-138-2-3p induced cell apoptosis after radiation. (A) and (B) show the comparison of early and late apoptosis between 100nM-TR and 100nMN-CR of Hep-2 cell line after radiation, respectively; (C) and (D) show the comparison of early and late apoptosis between 100nM-TR and 100nMN-CR of M2e cell line after radiation, respectively; (E) and (F) show the comparison of early and late apoptosis between 100nM-TR and 100nMN-CR of TU212 cell line after radiation, respectively. The vertical and horizontal axis stand for cell numbers and early/late apoptosis, respectively. We found that the cell numbers of early apoptosis and late apoptosis of Hep-2, M2e, and TU212 CSCs induced by transfection of 100nM hsa-miR-138-2-3p were larger than that induced by transfection of 100nM nonsense oligonucleotides, respectively, after radiation, the differences between them were statistically significant (*** P

    Journal: PeerJ

    Article Title: Radiosensitization effect of hsa-miR-138-2-3p on human laryngeal cancer stem cells

    doi: 10.7717/peerj.3233

    Figure Lengend Snippet: Overexpressed hsa-miR-138-2-3p induced cell apoptosis after radiation. (A) and (B) show the comparison of early and late apoptosis between 100nM-TR and 100nMN-CR of Hep-2 cell line after radiation, respectively; (C) and (D) show the comparison of early and late apoptosis between 100nM-TR and 100nMN-CR of M2e cell line after radiation, respectively; (E) and (F) show the comparison of early and late apoptosis between 100nM-TR and 100nMN-CR of TU212 cell line after radiation, respectively. The vertical and horizontal axis stand for cell numbers and early/late apoptosis, respectively. We found that the cell numbers of early apoptosis and late apoptosis of Hep-2, M2e, and TU212 CSCs induced by transfection of 100nM hsa-miR-138-2-3p were larger than that induced by transfection of 100nM nonsense oligonucleotides, respectively, after radiation, the differences between them were statistically significant (*** P

    Article Snippet: Laryngeal cancer sphere culture Three human laryngeal squamous cancer cell lines, Hep-2, TU212 and M2e, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

    Techniques: Transfection