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  • 99
    Thermo Fisher lipofectamine 2000
    Lipofectamine 2000, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 466214 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore bewo cell
    Increase of furin expression during <t>FSK-induced</t> syncytialization of choriocarcinoma <t>BeWo</t> cells. ( a ) BeWo cells were incubated in the medium with or without 100 μ M FSK (methanol as a vehicle control, also hereafter) for the indicated time before being subjected to protein extraction and western blotting with the indicated antibodies. Shown at the bottom are representative western blotting images. Three independent experiments were quantified by measuring the intensities of furin protein bands relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) controls (top) (** P
    Bewo Cell, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 78 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore t 84 cells
    Mouse serum in vitro antibody neutralizing activity against CT. Intracellular cyclic AMP levels in <t>T-84</t> cells measured with an EIA cAMP ELISA kit (Assay Design) showed anti-LT antibody neutralization activity. Neutralizing antibodies prevent CT from stimulating intracellular cAMP in T-84 cells, resulting in a low cAMP level (pmole/ml). The pooled serum sample of the mice immunized with ‘LT R19G -STb-Stx2e-3xSTa P12F ’ or the control mice (30 μl in total) was incubated with 10 ng CT toxin (in 150 μl) for 1 h at room temperature, the serum/toxin mixture was brought to 300 μl and added to T-84 cells (in 700 μl cell culture medium). Intracellular cAMP levels in cells were measured after 3 h incubation. Ten ng CT without serum was used as a positive control, and medium (cell culture medium; without toxin or serum) was used as the background reference. Columns and bars indicated mean cAMP levels and standard deviations. The p value of Mood’s Median test indicated difference of antibody neutralizing activity against CT between the immunization group and the control group.
    T 84 Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore sb203580
    MAPK14-activated proinflammatory gene program is p65/NF-κB-dependent. A) HCASMCs were transfected with si MAPK14 or siRNA control (si con ) for 72 h before RNA isolation for qRT-PCR of the indicated genes. B) HASMCs were transduced with Ad-MKK6 or equal amount of control virus (Ad-Empty) for 72 h followed by RNA isolation for qRT-PCR assessment of the indicated genes. C) HASMCs were pretreated with 10 μM of <t>SB203580</t> for 30 min prior to 4 ng/ml IL-1b stimulation for 24 h. RNA was isolated for the assessment of the indicated genes by qRT-PCR. D) HCASMCs were transfected with si MAPK14 or si con for 72 h followed by western blotting for the indicated proteins (Left) and its densitometric analysis of protein levels normalized to loading control TUBA (right). E) HASMCs were pretreated with 5 μM of Bay117082 for 45 min prior to Ad-MKK6 or control virus (Ad-empty) transduction for 48 h. RNA was isolated for qRT-PCR assessment of the indicated genes. Results were representative of at least 3 separate experiments and 3 biological replicates were included for each experiment. Data shown are mean ± SD. * P
    Sb203580, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 8906 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Millipore jak inhibitor 1
    MAPK14-activated proinflammatory gene program is p65/NF-κB-dependent. A) HCASMCs were transfected with si MAPK14 or siRNA control (si con ) for 72 h before RNA isolation for qRT-PCR of the indicated genes. B) HASMCs were transduced with Ad-MKK6 or equal amount of control virus (Ad-Empty) for 72 h followed by RNA isolation for qRT-PCR assessment of the indicated genes. C) HASMCs were pretreated with 10 μM of <t>SB203580</t> for 30 min prior to 4 ng/ml IL-1b stimulation for 24 h. RNA was isolated for the assessment of the indicated genes by qRT-PCR. D) HCASMCs were transfected with si MAPK14 or si con for 72 h followed by western blotting for the indicated proteins (Left) and its densitometric analysis of protein levels normalized to loading control TUBA (right). E) HASMCs were pretreated with 5 μM of Bay117082 for 45 min prior to Ad-MKK6 or control virus (Ad-empty) transduction for 48 h. RNA was isolated for qRT-PCR assessment of the indicated genes. Results were representative of at least 3 separate experiments and 3 biological replicates were included for each experiment. Data shown are mean ± SD. * P
    Jak Inhibitor 1, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 293 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore doxycycline
    MAPK14-activated proinflammatory gene program is p65/NF-κB-dependent. A) HCASMCs were transfected with si MAPK14 or siRNA control (si con ) for 72 h before RNA isolation for qRT-PCR of the indicated genes. B) HASMCs were transduced with Ad-MKK6 or equal amount of control virus (Ad-Empty) for 72 h followed by RNA isolation for qRT-PCR assessment of the indicated genes. C) HASMCs were pretreated with 10 μM of <t>SB203580</t> for 30 min prior to 4 ng/ml IL-1b stimulation for 24 h. RNA was isolated for the assessment of the indicated genes by qRT-PCR. D) HCASMCs were transfected with si MAPK14 or si con for 72 h followed by western blotting for the indicated proteins (Left) and its densitometric analysis of protein levels normalized to loading control TUBA (right). E) HASMCs were pretreated with 5 μM of Bay117082 for 45 min prior to Ad-MKK6 or control virus (Ad-empty) transduction for 48 h. RNA was isolated for qRT-PCR assessment of the indicated genes. Results were representative of at least 3 separate experiments and 3 biological replicates were included for each experiment. Data shown are mean ± SD. * P
    Doxycycline, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 13486 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore xav939
    MAPK14-activated proinflammatory gene program is p65/NF-κB-dependent. A) HCASMCs were transfected with si MAPK14 or siRNA control (si con ) for 72 h before RNA isolation for qRT-PCR of the indicated genes. B) HASMCs were transduced with Ad-MKK6 or equal amount of control virus (Ad-Empty) for 72 h followed by RNA isolation for qRT-PCR assessment of the indicated genes. C) HASMCs were pretreated with 10 μM of <t>SB203580</t> for 30 min prior to 4 ng/ml IL-1b stimulation for 24 h. RNA was isolated for the assessment of the indicated genes by qRT-PCR. D) HCASMCs were transfected with si MAPK14 or si con for 72 h followed by western blotting for the indicated proteins (Left) and its densitometric analysis of protein levels normalized to loading control TUBA (right). E) HASMCs were pretreated with 5 μM of Bay117082 for 45 min prior to Ad-MKK6 or control virus (Ad-empty) transduction for 48 h. RNA was isolated for qRT-PCR assessment of the indicated genes. Results were representative of at least 3 separate experiments and 3 biological replicates were included for each experiment. Data shown are mean ± SD. * P
    Xav939, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 570 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Immucor enzyme papain
    MAPK14-activated proinflammatory gene program is p65/NF-κB-dependent. A) HCASMCs were transfected with si MAPK14 or siRNA control (si con ) for 72 h before RNA isolation for qRT-PCR of the indicated genes. B) HASMCs were transduced with Ad-MKK6 or equal amount of control virus (Ad-Empty) for 72 h followed by RNA isolation for qRT-PCR assessment of the indicated genes. C) HASMCs were pretreated with 10 μM of <t>SB203580</t> for 30 min prior to 4 ng/ml IL-1b stimulation for 24 h. RNA was isolated for the assessment of the indicated genes by qRT-PCR. D) HCASMCs were transfected with si MAPK14 or si con for 72 h followed by western blotting for the indicated proteins (Left) and its densitometric analysis of protein levels normalized to loading control TUBA (right). E) HASMCs were pretreated with 5 μM of Bay117082 for 45 min prior to Ad-MKK6 or control virus (Ad-empty) transduction for 48 h. RNA was isolated for qRT-PCR assessment of the indicated genes. Results were representative of at least 3 separate experiments and 3 biological replicates were included for each experiment. Data shown are mean ± SD. * P
    Enzyme Papain, supplied by Immucor, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore sb202190
    <t>SB202190</t> enhances cleavage of PARP when combined with glucose analogs. Western blot analysis of whole cell lysates from cells treated for 24 hours with 2-DG or D-allose alone or in combination with SB202190. Lysates were probed for cleaved and total PARP. Tubulin served as a loading control for protein. Cleaved PARP band density was quantified using ImageJ software with normalization to α-Tubulin. One representative experiment is shown, n = 3.
    Sb202190, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2487 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore celecoxib
    Protein expression of p-ERK and p-CREB (A) or p-AKT and p-Src (B) in BxPC-3 xenografts of stress free (NS) and social stress (SS) exposed mice. Western blots (A and B) illustrating induction of p-ERK, p-CREB, p-AKT and p-Src by social stress and inhibition of these responses by celcoxib alone or <t>celecoxib</t> + GABA. Quantitative assessment of these changes at the protein level were determined in the ELISA assays shown in Figure C and D. Induction of all investigated phosphorylated proteins by social stress (SS) was significant ( p
    Celecoxib, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 744 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore adenosine diphosphate
    Protein expression of p-ERK and p-CREB (A) or p-AKT and p-Src (B) in BxPC-3 xenografts of stress free (NS) and social stress (SS) exposed mice. Western blots (A and B) illustrating induction of p-ERK, p-CREB, p-AKT and p-Src by social stress and inhibition of these responses by celcoxib alone or <t>celecoxib</t> + GABA. Quantitative assessment of these changes at the protein level were determined in the ELISA assays shown in Figure C and D. Induction of all investigated phosphorylated proteins by social stress (SS) was significant ( p
    Adenosine Diphosphate, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 261 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher antisense mirna mimics
    Protein expression of p-ERK and p-CREB (A) or p-AKT and p-Src (B) in BxPC-3 xenografts of stress free (NS) and social stress (SS) exposed mice. Western blots (A and B) illustrating induction of p-ERK, p-CREB, p-AKT and p-Src by social stress and inhibition of these responses by celcoxib alone or <t>celecoxib</t> + GABA. Quantitative assessment of these changes at the protein level were determined in the ELISA assays shown in Figure C and D. Induction of all investigated phosphorylated proteins by social stress (SS) was significant ( p
    Antisense Mirna Mimics, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore curcumin
    Protein expression of p-ERK and p-CREB (A) or p-AKT and p-Src (B) in BxPC-3 xenografts of stress free (NS) and social stress (SS) exposed mice. Western blots (A and B) illustrating induction of p-ERK, p-CREB, p-AKT and p-Src by social stress and inhibition of these responses by celcoxib alone or <t>celecoxib</t> + GABA. Quantitative assessment of these changes at the protein level were determined in the ELISA assays shown in Figure C and D. Induction of all investigated phosphorylated proteins by social stress (SS) was significant ( p
    Curcumin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2184 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore mitomycin c
    Effect of fibulin 3 knock-down on migration of HCT116 cells. HCT116 cells with a permanent (shFib3) or transient (sh2Fib3) fibulin 3 knock-down using two different human fibulin 3 shRNAs were used. A and C , Western blot analysis of Fibulin 3 protein levels in the culture medium from HCT116 cells (non-silenced (−) and p38α knock-down (shp38α), with (shFib3 (in A ) and sh2Fib3 (in C )) or without fibulin 3 knock-down) normalized with β-actin (in cell extracts) and its quantification. p38α was used as a control of its expression. B and D , wound healing assay. Cells were pretreated with <t>mitomycin</t> and maintained with 2% serum, with or without 40 ng/ml of HGF (control), as indicated. Left panels , representative images from phase contrast microscope at 0 and 72 h (in B ) or at 0 and 48 h (in D ) of migration. Right panels , histograms showing the mean value ± S.E. of the percentage of wound closure at 24, 48, and 72 h (in B ) or at 48 h (in D ) with or without HGF ( n = 4), as indicated. *, p
    Mitomycin C, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 11777 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore su11274
    Effect of MET and EGFR inhibition on heat stress and/or growth factor induced MET and EGFR signaling in HCC cells. (A) N1S1 and (B) AS30D HCC cells were treated with a dose-titration of the MET inhibitor <t>SU11274</t> (0.04μM-10μM), EGFR inhibitor erlotinib (0.08μM-20μM) or vehicle control (0.1% DMSO) and assessed for viability at 72 hours using WST-1 assay. Data were normalized to vehicle control and the IC 50 estimated using non-linear regression curve fitting. Data are presented as mean±SEM of 3 independent experiments. (C) N1S1 and (D) AS30D HCC cells pre-treated with SU11274 (1μM and 10μM), erlotinib (1μM and 10μM) or vehicle control (0.1% DMSO) for 1 hour, heat stressed (45°C) or control (37°C) for 10 minutes, harvested immediately post-heat stress and whole-cell lysates were subjected to Western blotting using phospho-specific and total antibodies against MET and EGFR. β-actin was used as a loading control. (E) N1S1 cells pre-treated for 1 hour with SU11274 (5μM) or vehicle control (0.1% DMSO) followed by heat stress (45°C) or control (37°C) ± concomitant treatment with recombinant HGF (50ng/ml) or vehicle control for 10 minutes. Immediately post-heat stress whole-cell lysates were subjected to Western blotting for phospho- and total MET. β-actin was used as a loading control. (F) AS30D cells pre-treated for 1 hour with erlotinib (5μM) or vehicle control (0.1% DMSO) followed by heat stress (45°C) or control (37°C) ± concomitant treatment with recombinant EGF (50ng/ml or vehicle control for 10 minutes. Immediately post-heat stress whole-cell lysates were subjected to Western blotting for phospho- and total EGFR. β-actin was used as a loading control.
    Su11274, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 287 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Increase of furin expression during FSK-induced syncytialization of choriocarcinoma BeWo cells. ( a ) BeWo cells were incubated in the medium with or without 100 μ M FSK (methanol as a vehicle control, also hereafter) for the indicated time before being subjected to protein extraction and western blotting with the indicated antibodies. Shown at the bottom are representative western blotting images. Three independent experiments were quantified by measuring the intensities of furin protein bands relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) controls (top) (** P

    Journal: Cell Death & Disease

    Article Title: The proprotein convertase furin is required for trophoblast syncytialization

    doi: 10.1038/cddis.2013.106

    Figure Lengend Snippet: Increase of furin expression during FSK-induced syncytialization of choriocarcinoma BeWo cells. ( a ) BeWo cells were incubated in the medium with or without 100 μ M FSK (methanol as a vehicle control, also hereafter) for the indicated time before being subjected to protein extraction and western blotting with the indicated antibodies. Shown at the bottom are representative western blotting images. Three independent experiments were quantified by measuring the intensities of furin protein bands relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) controls (top) (** P

    Article Snippet: Cell fusion was induced by treating BeWo cell with FSK (Sigma-Aldrich, St. Louis, MO, USA).

    Techniques: Expressing, Incubation, Protein Extraction, Western Blot

    Furin is required for FSK-induced syncytialization of choriocarcinoma BeWo cells. ( a and b ) BeWo cells treated with control (Con) small interfering RNA (siRNA) or furin siRNA were incubated in the medium with or without FSK for 48 h. ( a ) Cells were lysed and the resulting extracts were subjected to western blotting with the indicated antibodies. ( b ) Cells were stained with 4',6-diamidino-2-phenylindole (DAPI) (blue) and anti-E-cadherin (green). Shown is a typical image of mononucleated (red arrow) or multinucleated (white arrow) cells (left panel). Bar=20 μ m. Three independent experiments were quantified by analyzing the ratio of multinucleated cells in different treatment groups (right panel) (** P

    Journal: Cell Death & Disease

    Article Title: The proprotein convertase furin is required for trophoblast syncytialization

    doi: 10.1038/cddis.2013.106

    Figure Lengend Snippet: Furin is required for FSK-induced syncytialization of choriocarcinoma BeWo cells. ( a and b ) BeWo cells treated with control (Con) small interfering RNA (siRNA) or furin siRNA were incubated in the medium with or without FSK for 48 h. ( a ) Cells were lysed and the resulting extracts were subjected to western blotting with the indicated antibodies. ( b ) Cells were stained with 4',6-diamidino-2-phenylindole (DAPI) (blue) and anti-E-cadherin (green). Shown is a typical image of mononucleated (red arrow) or multinucleated (white arrow) cells (left panel). Bar=20 μ m. Three independent experiments were quantified by analyzing the ratio of multinucleated cells in different treatment groups (right panel) (** P

    Article Snippet: Cell fusion was induced by treating BeWo cell with FSK (Sigma-Aldrich, St. Louis, MO, USA).

    Techniques: Small Interfering RNA, Incubation, Western Blot, Staining

    Mouse serum in vitro antibody neutralizing activity against CT. Intracellular cyclic AMP levels in T-84 cells measured with an EIA cAMP ELISA kit (Assay Design) showed anti-LT antibody neutralization activity. Neutralizing antibodies prevent CT from stimulating intracellular cAMP in T-84 cells, resulting in a low cAMP level (pmole/ml). The pooled serum sample of the mice immunized with ‘LT R19G -STb-Stx2e-3xSTa P12F ’ or the control mice (30 μl in total) was incubated with 10 ng CT toxin (in 150 μl) for 1 h at room temperature, the serum/toxin mixture was brought to 300 μl and added to T-84 cells (in 700 μl cell culture medium). Intracellular cAMP levels in cells were measured after 3 h incubation. Ten ng CT without serum was used as a positive control, and medium (cell culture medium; without toxin or serum) was used as the background reference. Columns and bars indicated mean cAMP levels and standard deviations. The p value of Mood’s Median test indicated difference of antibody neutralizing activity against CT between the immunization group and the control group.

    Journal: Veterinary Microbiology

    Article Title: Antibodies derived from a toxoid MEFA (multiepitope fusion antigen) show neutralizing activities against heat-labile toxin (LT), heat-stable toxins (STa, STb), and Shiga toxin 2e (Stx2e) of porcine enterotoxigenic Escherichia coli (ETEC)

    doi: 10.1016/j.vetmic.2016.02.002

    Figure Lengend Snippet: Mouse serum in vitro antibody neutralizing activity against CT. Intracellular cyclic AMP levels in T-84 cells measured with an EIA cAMP ELISA kit (Assay Design) showed anti-LT antibody neutralization activity. Neutralizing antibodies prevent CT from stimulating intracellular cAMP in T-84 cells, resulting in a low cAMP level (pmole/ml). The pooled serum sample of the mice immunized with ‘LT R19G -STb-Stx2e-3xSTa P12F ’ or the control mice (30 μl in total) was incubated with 10 ng CT toxin (in 150 μl) for 1 h at room temperature, the serum/toxin mixture was brought to 300 μl and added to T-84 cells (in 700 μl cell culture medium). Intracellular cAMP levels in cells were measured after 3 h incubation. Ten ng CT without serum was used as a positive control, and medium (cell culture medium; without toxin or serum) was used as the background reference. Columns and bars indicated mean cAMP levels and standard deviations. The p value of Mood’s Median test indicated difference of antibody neutralizing activity against CT between the immunization group and the control group.

    Article Snippet: Incubated serum/toxin mixture was brought to 300 μl with DMEM/F-12 and added to a confluent monolayer of T-84 cells (105 cells per well, in 700 μl DMEM/F-12) pre-treated with 1 mM IBMX (3-isobutyl-1-methylaxanthine; Sigma).

    Techniques: In Vitro, Activity Assay, Enzyme-linked Immunosorbent Assay, Neutralization, Mouse Assay, Incubation, Cell Culture, Positive Control

    Mouse serum in vitro antibody neutralizing activity against STa toxin. Intracellular cyclic GMP levels in T-84 cells measured with an EIA cGMP ELISA kit (Assay Design) showed anti-STa antibody neutralization activity. Neutralizing antibodies prevent STa toxin from stimulating intracellular cGMP in T-84 cells, resulting in a low cGMP level (pmole/ml). The pooled serum sample of the mice immunized with ‘LT R19G -STb-Stx2e-3xSTa P12F ’ or the control mice (30 μl in total) was incubated with 2 ng STa toxin (in 150 μl) for 1 h at room temperature, the serum/toxin mixture was brought to 300 μl and added to T-84 cells (in 700 μl cell culture medium). Intracellular cGMP levels in cells were measured after 1 h incubation. Two ng STa without serum was used as a positive control of enterotoxicity and medium (cell culture medium; without toxin or serum) was used as the background reference. Columns and bars indicated mean cGMP levels and standard deviations. The p value of Mood’s Median test indicated difference of antibody neutralizing activity against STa between the immunization group and the control group.

    Journal: Veterinary Microbiology

    Article Title: Antibodies derived from a toxoid MEFA (multiepitope fusion antigen) show neutralizing activities against heat-labile toxin (LT), heat-stable toxins (STa, STb), and Shiga toxin 2e (Stx2e) of porcine enterotoxigenic Escherichia coli (ETEC)

    doi: 10.1016/j.vetmic.2016.02.002

    Figure Lengend Snippet: Mouse serum in vitro antibody neutralizing activity against STa toxin. Intracellular cyclic GMP levels in T-84 cells measured with an EIA cGMP ELISA kit (Assay Design) showed anti-STa antibody neutralization activity. Neutralizing antibodies prevent STa toxin from stimulating intracellular cGMP in T-84 cells, resulting in a low cGMP level (pmole/ml). The pooled serum sample of the mice immunized with ‘LT R19G -STb-Stx2e-3xSTa P12F ’ or the control mice (30 μl in total) was incubated with 2 ng STa toxin (in 150 μl) for 1 h at room temperature, the serum/toxin mixture was brought to 300 μl and added to T-84 cells (in 700 μl cell culture medium). Intracellular cGMP levels in cells were measured after 1 h incubation. Two ng STa without serum was used as a positive control of enterotoxicity and medium (cell culture medium; without toxin or serum) was used as the background reference. Columns and bars indicated mean cGMP levels and standard deviations. The p value of Mood’s Median test indicated difference of antibody neutralizing activity against STa between the immunization group and the control group.

    Article Snippet: Incubated serum/toxin mixture was brought to 300 μl with DMEM/F-12 and added to a confluent monolayer of T-84 cells (105 cells per well, in 700 μl DMEM/F-12) pre-treated with 1 mM IBMX (3-isobutyl-1-methylaxanthine; Sigma).

    Techniques: In Vitro, Activity Assay, Enzyme-linked Immunosorbent Assay, Neutralization, Mouse Assay, Incubation, Cell Culture, Positive Control

    MAPK14-activated proinflammatory gene program is p65/NF-κB-dependent. A) HCASMCs were transfected with si MAPK14 or siRNA control (si con ) for 72 h before RNA isolation for qRT-PCR of the indicated genes. B) HASMCs were transduced with Ad-MKK6 or equal amount of control virus (Ad-Empty) for 72 h followed by RNA isolation for qRT-PCR assessment of the indicated genes. C) HASMCs were pretreated with 10 μM of SB203580 for 30 min prior to 4 ng/ml IL-1b stimulation for 24 h. RNA was isolated for the assessment of the indicated genes by qRT-PCR. D) HCASMCs were transfected with si MAPK14 or si con for 72 h followed by western blotting for the indicated proteins (Left) and its densitometric analysis of protein levels normalized to loading control TUBA (right). E) HASMCs were pretreated with 5 μM of Bay117082 for 45 min prior to Ad-MKK6 or control virus (Ad-empty) transduction for 48 h. RNA was isolated for qRT-PCR assessment of the indicated genes. Results were representative of at least 3 separate experiments and 3 biological replicates were included for each experiment. Data shown are mean ± SD. * P

    Journal: Redox Biology

    Article Title: Vascular smooth muscle-MAPK14 is required for neointimal hyperplasia by suppressing VSMC differentiation and inducing proliferation and inflammation

    doi: 10.1016/j.redox.2019.101137

    Figure Lengend Snippet: MAPK14-activated proinflammatory gene program is p65/NF-κB-dependent. A) HCASMCs were transfected with si MAPK14 or siRNA control (si con ) for 72 h before RNA isolation for qRT-PCR of the indicated genes. B) HASMCs were transduced with Ad-MKK6 or equal amount of control virus (Ad-Empty) for 72 h followed by RNA isolation for qRT-PCR assessment of the indicated genes. C) HASMCs were pretreated with 10 μM of SB203580 for 30 min prior to 4 ng/ml IL-1b stimulation for 24 h. RNA was isolated for the assessment of the indicated genes by qRT-PCR. D) HCASMCs were transfected with si MAPK14 or si con for 72 h followed by western blotting for the indicated proteins (Left) and its densitometric analysis of protein levels normalized to loading control TUBA (right). E) HASMCs were pretreated with 5 μM of Bay117082 for 45 min prior to Ad-MKK6 or control virus (Ad-empty) transduction for 48 h. RNA was isolated for qRT-PCR assessment of the indicated genes. Results were representative of at least 3 separate experiments and 3 biological replicates were included for each experiment. Data shown are mean ± SD. * P

    Article Snippet: For inhibitor treatment, serum starved VSMCs were pretreated with either SB203580 (Calbiochem, CAS 869185-85-3) or Bay117082 (Selleckchem No. S2913) at a dose of 5 µM for 45 min followed by PDGF (25 ng/ml, R & D, #220-BB-010) induction for 48 h prior to cell counting, or IL1β (4 ng/ml, R & D, #201-LB) for 24 h before RNA isolation.

    Techniques: Transfection, Isolation, Quantitative RT-PCR, Transduction, Western Blot

    Deficiency of MAPK14 in VSMCs suppresses VSMC proliferation. A) Representative photomicrographs of immunofluorescence staining for a proliferation marker, Ki67 in carotid arteries isolated at 2 weeks after ligation from MAPK14 +/+ and iSMC-MAPK14 -/- mice (Left). The percentage of Ki67 + cells in injured carotid arteries was shown (right). Data shown are mean ± SD, n = 5. Scale bar: 20 µm. B) HASMCs were starved overnight followed by treatment with 5 μM SB203580 or vehicle control for 30 min prior to stimulation with 50 ng/ml PDGF. Cells were counted by hemocytometer at 24 h after PDGF stimulation. C) Growing HASMCs were transduced with adenovirus carrying a constitutively active form of MKK6 (Ad-MKK6) or equal amount of control adenovirus (Ad-Empty) for 72 h before cell counting as above. Cell proliferation was defined as fold change compared to vehicle control (set to 1). D) HCASMs were transfected with si MAPK14 or siRNA control (si con ) for 72 h before RNA isolation for qRT-PCR of the indicated genes. Data shown in B–D are mean ± SD and are representative of 3 separate experiments. * P

    Journal: Redox Biology

    Article Title: Vascular smooth muscle-MAPK14 is required for neointimal hyperplasia by suppressing VSMC differentiation and inducing proliferation and inflammation

    doi: 10.1016/j.redox.2019.101137

    Figure Lengend Snippet: Deficiency of MAPK14 in VSMCs suppresses VSMC proliferation. A) Representative photomicrographs of immunofluorescence staining for a proliferation marker, Ki67 in carotid arteries isolated at 2 weeks after ligation from MAPK14 +/+ and iSMC-MAPK14 -/- mice (Left). The percentage of Ki67 + cells in injured carotid arteries was shown (right). Data shown are mean ± SD, n = 5. Scale bar: 20 µm. B) HASMCs were starved overnight followed by treatment with 5 μM SB203580 or vehicle control for 30 min prior to stimulation with 50 ng/ml PDGF. Cells were counted by hemocytometer at 24 h after PDGF stimulation. C) Growing HASMCs were transduced with adenovirus carrying a constitutively active form of MKK6 (Ad-MKK6) or equal amount of control adenovirus (Ad-Empty) for 72 h before cell counting as above. Cell proliferation was defined as fold change compared to vehicle control (set to 1). D) HCASMs were transfected with si MAPK14 or siRNA control (si con ) for 72 h before RNA isolation for qRT-PCR of the indicated genes. Data shown in B–D are mean ± SD and are representative of 3 separate experiments. * P

    Article Snippet: For inhibitor treatment, serum starved VSMCs were pretreated with either SB203580 (Calbiochem, CAS 869185-85-3) or Bay117082 (Selleckchem No. S2913) at a dose of 5 µM for 45 min followed by PDGF (25 ng/ml, R & D, #220-BB-010) induction for 48 h prior to cell counting, or IL1β (4 ng/ml, R & D, #201-LB) for 24 h before RNA isolation.

    Techniques: Immunofluorescence, Staining, Marker, Isolation, Ligation, Mouse Assay, Transduction, Cell Counting, Transfection, Quantitative RT-PCR

    p38 and JNK MAP kinase inhibition decreases apoptosis induced by iso NH 2 CA-4 in EBV-infected BL41B95.8 cells. Cells were treated with 10 nM iso NH 2 CA-4 with or without 1 h pretreatment with the p38 inhibitor SB203580 (25 μM), the JNK inhibitor SP600125 (7.5 nM) or both. ( a and b ) An example of apoptosis induction and mitochondrial loss of integrity in BL41B95.8 cells as assessed on flow cytometry biparametric histogram after annexin-V and propidium iodide ( a ) or DiOC6(3) and propidium iodide ( b ) labeling. Percentages of apoptotic cells are indicated in each graph. ( c ) Percentages of BL2B95.8 (left panel) and BL41.B95.8 (right panel) annexin-V-positive cells in presence (+) or absence (−) of iso NH 2 CA-4 and/or both p38 and JNK inhibitors at 24 and 48 h. Results are representative of three independent experiments

    Journal: Cell Death & Disease

    Article Title: Reversion of apoptotic resistance of TP53-mutated Burkitt lymphoma B-cells to spindle poisons by exogenous activation of JNK and p38 MAP kinases

    doi: 10.1038/cddis.2014.150

    Figure Lengend Snippet: p38 and JNK MAP kinase inhibition decreases apoptosis induced by iso NH 2 CA-4 in EBV-infected BL41B95.8 cells. Cells were treated with 10 nM iso NH 2 CA-4 with or without 1 h pretreatment with the p38 inhibitor SB203580 (25 μM), the JNK inhibitor SP600125 (7.5 nM) or both. ( a and b ) An example of apoptosis induction and mitochondrial loss of integrity in BL41B95.8 cells as assessed on flow cytometry biparametric histogram after annexin-V and propidium iodide ( a ) or DiOC6(3) and propidium iodide ( b ) labeling. Percentages of apoptotic cells are indicated in each graph. ( c ) Percentages of BL2B95.8 (left panel) and BL41.B95.8 (right panel) annexin-V-positive cells in presence (+) or absence (−) of iso NH 2 CA-4 and/or both p38 and JNK inhibitors at 24 and 48 h. Results are representative of three independent experiments

    Article Snippet: Cells were pretreated or not with the p38 inhibitor SB203580 (25 μ M) (Calbiochem, VWR, Strasbourg, France), the JNK inhibitor SP600125 (7.5 nM) (Sigma-Aldrich, Saint Quentin Fallavier, France) or D-erythro-dihydrosphingosine (7.5 μ M) (Sigma-Aldrich) for 1 h. All drugs were reconstituted in dimethyl sulfoxide (DMSO), used as control treatment.

    Techniques: Inhibition, Infection, Flow Cytometry, Cytometry, Labeling

    p38 and JNK inhibitors counteract apoptosis induced by any class of spindle poisons in EBV-positive BL41B95.8 cells. BL41 and BL41B95.8 cells were treated with actin-stabilizing (Taxol) or with actin-destabilizing drugs specific for the colchicine site (colchicine: 30 nM Col; 10 nM ( iso NH 2 CA-4, isoCA-4); 20 nM CA-4) or the vinca site (vinblastine: 25 nM Vinb; vincristine: 25 nM Vinc) for 24 h. Cells were additionally pretreated (+) or not (−) for 1 h with the p38 inhibitor SB203580 (25 μ M) and the JNK inhibitor SP600125 (7.5 nM). ( a ) Percentages of BL41 and BL41B95.8 annexin-V-positive cells assessed by flow cytometry. ( b ) Percentages of DiOC6(3) negative cells, assessed by flow cytometry. These results correspond to at least three independent experiments

    Journal: Cell Death & Disease

    Article Title: Reversion of apoptotic resistance of TP53-mutated Burkitt lymphoma B-cells to spindle poisons by exogenous activation of JNK and p38 MAP kinases

    doi: 10.1038/cddis.2014.150

    Figure Lengend Snippet: p38 and JNK inhibitors counteract apoptosis induced by any class of spindle poisons in EBV-positive BL41B95.8 cells. BL41 and BL41B95.8 cells were treated with actin-stabilizing (Taxol) or with actin-destabilizing drugs specific for the colchicine site (colchicine: 30 nM Col; 10 nM ( iso NH 2 CA-4, isoCA-4); 20 nM CA-4) or the vinca site (vinblastine: 25 nM Vinb; vincristine: 25 nM Vinc) for 24 h. Cells were additionally pretreated (+) or not (−) for 1 h with the p38 inhibitor SB203580 (25 μ M) and the JNK inhibitor SP600125 (7.5 nM). ( a ) Percentages of BL41 and BL41B95.8 annexin-V-positive cells assessed by flow cytometry. ( b ) Percentages of DiOC6(3) negative cells, assessed by flow cytometry. These results correspond to at least three independent experiments

    Article Snippet: Cells were pretreated or not with the p38 inhibitor SB203580 (25 μ M) (Calbiochem, VWR, Strasbourg, France), the JNK inhibitor SP600125 (7.5 nM) (Sigma-Aldrich, Saint Quentin Fallavier, France) or D-erythro-dihydrosphingosine (7.5 μ M) (Sigma-Aldrich) for 1 h. All drugs were reconstituted in dimethyl sulfoxide (DMSO), used as control treatment.

    Techniques: Flow Cytometry, Cytometry

    A selective p38 pathway inhibitor SB203580 (1 and 10 μM) could significantly suppress LPS-induced MCP-1 production for 24 h (A) and 48 h (B) in THP-1 cells. A selective vitamin D receptor blocking antibody could reverse the suppressive effect

    Journal: Acta Cardiologica Sinica

    Article Title: Effect of Vitamin D3 on Monocyte Chemoattractant Protein 1 Production in Monocytes and Macrophages

    doi:

    Figure Lengend Snippet: A selective p38 pathway inhibitor SB203580 (1 and 10 μM) could significantly suppress LPS-induced MCP-1 production for 24 h (A) and 48 h (B) in THP-1 cells. A selective vitamin D receptor blocking antibody could reverse the suppressive effect

    Article Snippet: In some cases, THP-1 cells were pre-treated with a selective p38 pathway inhibitor SB203580 or vitamin D receptor blocking the antibody one hour before 1α,25-(OH)2D3 (Sigma Chemical Co., St. Louis, MO, USA).

    Techniques: Blocking Assay

    Role of spinal p38 activation in CCL3- and carrageenan-induced chronic hyperalgesia (a) LPS-induced TNF-α production by LysM-GRK2 +/+ WT and LysM-GRK2 f/+ microglia. Primary cultures of WT and GRK2 +/− microglia were stimulated for 3 h with LPS in presence or absence of the p38 inhibitor SB203580 (20 μM). TNF-α in the supernatant was determined by ELISA. TNF-α levels were undetectable in unstimulated microglia. Data are from two independent experiments performed in triplicate. (b) Western blot analysis of p-p38 levels in lumbar spinal cord of saline or carrageenan-treated WT and GRK2 +/− mice at 7 days after intraplantar injection. (c) WT and GRK2 +/− mice were treated with an intrathecal injection of the p38 inhibitor SB239063 (5 μg/mouse) at day 7 after intraplantar carrageenan injection. Heat withdrawal latencies were determined 1 hour prior to and 2 hours after administration of SB239063 (n = 4). (d) WT and GRK2 +/− were treated with an intrathecal injection of Etanercept (100 μg/mouse) at day 7 after intraplantar carrageenan injection and heat withdrawal latencies were determined (n = 6). (e) TNF-α levels in spinal segments L1–L5 (lumbar) or T1–T8 (thoracic) of GRK2 +/− mice were determined 7 days after intraplantar vehicle or carrageenan injection. The effect of p38 inhibition on carrageenan-induced TNF-α was tested 7 days after intraplantar carrageenan by treating mice intrathecally with SB239063 24 h and 2 h prior to collection of spinal cords. Data represent the level of TNF-α expressed as percentage of vehicle-treated controls. Data are expressed as mean ± SEM. * p

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    Article Title: GRK2: a Novel Cell Specific Regulator of Severity and Duration of Inflammatory Pain

    doi: 10.1523/JNEUROSCI.5752-09.2010

    Figure Lengend Snippet: Role of spinal p38 activation in CCL3- and carrageenan-induced chronic hyperalgesia (a) LPS-induced TNF-α production by LysM-GRK2 +/+ WT and LysM-GRK2 f/+ microglia. Primary cultures of WT and GRK2 +/− microglia were stimulated for 3 h with LPS in presence or absence of the p38 inhibitor SB203580 (20 μM). TNF-α in the supernatant was determined by ELISA. TNF-α levels were undetectable in unstimulated microglia. Data are from two independent experiments performed in triplicate. (b) Western blot analysis of p-p38 levels in lumbar spinal cord of saline or carrageenan-treated WT and GRK2 +/− mice at 7 days after intraplantar injection. (c) WT and GRK2 +/− mice were treated with an intrathecal injection of the p38 inhibitor SB239063 (5 μg/mouse) at day 7 after intraplantar carrageenan injection. Heat withdrawal latencies were determined 1 hour prior to and 2 hours after administration of SB239063 (n = 4). (d) WT and GRK2 +/− were treated with an intrathecal injection of Etanercept (100 μg/mouse) at day 7 after intraplantar carrageenan injection and heat withdrawal latencies were determined (n = 6). (e) TNF-α levels in spinal segments L1–L5 (lumbar) or T1–T8 (thoracic) of GRK2 +/− mice were determined 7 days after intraplantar vehicle or carrageenan injection. The effect of p38 inhibition on carrageenan-induced TNF-α was tested 7 days after intraplantar carrageenan by treating mice intrathecally with SB239063 24 h and 2 h prior to collection of spinal cords. Data represent the level of TNF-α expressed as percentage of vehicle-treated controls. Data are expressed as mean ± SEM. * p

    Article Snippet: Plated microglia were pre-incubated for 1h with p38 inhibitors SB203580 (20 μM) (Sigma-Aldrich) before stimulation with 10 ng/ml LPS (Sigma-Aldrich).

    Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Mouse Assay, Injection, Inhibition

    p38 MAPK is required for efficient removal of CPD. A , inhibition of p38 MAPK activation by SB203580. OSU-2 cells were pretreated with DMSO or SB203580 (10 μ m ) for 30 min. The cells were then UV-irradiated at 10 J/m 2 and further cultured in

    Journal:

    Article Title: The p38 Mitogen-activated Protein Kinase Augments Nucleotide Excision Repair by Mediating DDB2 Degradation and Chromatin Relaxation *

    doi: 10.1074/jbc.M803963200

    Figure Lengend Snippet: p38 MAPK is required for efficient removal of CPD. A , inhibition of p38 MAPK activation by SB203580. OSU-2 cells were pretreated with DMSO or SB203580 (10 μ m ) for 30 min. The cells were then UV-irradiated at 10 J/m 2 and further cultured in

    Article Snippet: To study the function of p38 MAPK, the cells were pretreated with p38 MAPK inhibitor SB203580 (Calbiochem, San Diego, CA) or DMSO for 30 min before UV irradiation.

    Techniques: Inhibition, Activation Assay, Irradiation, Cell Culture

    p38 MAPK is involved in UV-induced chromatin relaxation. A , cell cycle distribution. OSU-2 cells grown in complete medium ( top panel ) and serum free medium ( bottom panel ) were analyzed by flow cytometry for cell cycle distribution. B , MNase sensitivity

    Journal:

    Article Title: The p38 Mitogen-activated Protein Kinase Augments Nucleotide Excision Repair by Mediating DDB2 Degradation and Chromatin Relaxation *

    doi: 10.1074/jbc.M803963200

    Figure Lengend Snippet: p38 MAPK is involved in UV-induced chromatin relaxation. A , cell cycle distribution. OSU-2 cells grown in complete medium ( top panel ) and serum free medium ( bottom panel ) were analyzed by flow cytometry for cell cycle distribution. B , MNase sensitivity

    Article Snippet: To study the function of p38 MAPK, the cells were pretreated with p38 MAPK inhibitor SB203580 (Calbiochem, San Diego, CA) or DMSO for 30 min before UV irradiation.

    Techniques: Flow Cytometry, Cytometry

    p38 MAPK affects the recruitment of XPC and XPB to CPD foci. A and B , recruitment of XPC and XPB to CPD. OSU-2 cells were grown on coverslips and pretreated with DMSO or SB203580 for 30 min prior to UV irradiation (100 J/m 2 ) through a 5-μm

    Journal:

    Article Title: The p38 Mitogen-activated Protein Kinase Augments Nucleotide Excision Repair by Mediating DDB2 Degradation and Chromatin Relaxation *

    doi: 10.1074/jbc.M803963200

    Figure Lengend Snippet: p38 MAPK affects the recruitment of XPC and XPB to CPD foci. A and B , recruitment of XPC and XPB to CPD. OSU-2 cells were grown on coverslips and pretreated with DMSO or SB203580 for 30 min prior to UV irradiation (100 J/m 2 ) through a 5-μm

    Article Snippet: To study the function of p38 MAPK, the cells were pretreated with p38 MAPK inhibitor SB203580 (Calbiochem, San Diego, CA) or DMSO for 30 min before UV irradiation.

    Techniques: Irradiation

    p38 MAPK mediates UV-induced DDB2 ubiquitylation and degradation. A and B , OSU-2 ( A ) or HeLa-DDB2 ( B ) cells were treated with DMSO or SB203580 for 30 min, UV-irradiated at 20 J/m 2 , and further incubated for indicated times. The whole cell extracts

    Journal:

    Article Title: The p38 Mitogen-activated Protein Kinase Augments Nucleotide Excision Repair by Mediating DDB2 Degradation and Chromatin Relaxation *

    doi: 10.1074/jbc.M803963200

    Figure Lengend Snippet: p38 MAPK mediates UV-induced DDB2 ubiquitylation and degradation. A and B , OSU-2 ( A ) or HeLa-DDB2 ( B ) cells were treated with DMSO or SB203580 for 30 min, UV-irradiated at 20 J/m 2 , and further incubated for indicated times. The whole cell extracts

    Article Snippet: To study the function of p38 MAPK, the cells were pretreated with p38 MAPK inhibitor SB203580 (Calbiochem, San Diego, CA) or DMSO for 30 min before UV irradiation.

    Techniques: Irradiation, Incubation

    p38 MAPK inhibitor SB203580 inhibits the activation of p38 MAPK but not JNK, ERK, or Akt in response to UV irradiation. A , chemical structure of SB203580. B and C , inhibition of p38 MAPK kinase activity by different doses of SB203580. OSU-2 cells (

    Journal:

    Article Title: The p38 Mitogen-activated Protein Kinase Augments Nucleotide Excision Repair by Mediating DDB2 Degradation and Chromatin Relaxation *

    doi: 10.1074/jbc.M803963200

    Figure Lengend Snippet: p38 MAPK inhibitor SB203580 inhibits the activation of p38 MAPK but not JNK, ERK, or Akt in response to UV irradiation. A , chemical structure of SB203580. B and C , inhibition of p38 MAPK kinase activity by different doses of SB203580. OSU-2 cells (

    Article Snippet: To study the function of p38 MAPK, the cells were pretreated with p38 MAPK inhibitor SB203580 (Calbiochem, San Diego, CA) or DMSO for 30 min before UV irradiation.

    Techniques: Activation Assay, Irradiation, Inhibition, Activity Assay

    Proposed model for positive modulation of NER by p38 MAPK activation following UV irradiation of human cells.

    Journal:

    Article Title: The p38 Mitogen-activated Protein Kinase Augments Nucleotide Excision Repair by Mediating DDB2 Degradation and Chromatin Relaxation *

    doi: 10.1074/jbc.M803963200

    Figure Lengend Snippet: Proposed model for positive modulation of NER by p38 MAPK activation following UV irradiation of human cells.

    Article Snippet: To study the function of p38 MAPK, the cells were pretreated with p38 MAPK inhibitor SB203580 (Calbiochem, San Diego, CA) or DMSO for 30 min before UV irradiation.

    Techniques: Activation Assay, Irradiation

    Inhibition of NF κ B and concomitant activation of JNK during genotoxic stress are critical for inducing ‘resistance to apoptosis' switchover in drug-resistant cancer cells. ( a ) Western blotting and confocal imaging experiments were conducted to determine nuclear translocation of p65NF κ B in cisplatin-treated A549 and MCF-7 cells ( b ) Cisplatin-treated, p65NF κ B-over-expressed MCF-7 cells and I κ B α -SR-constituted A549 cells were subjected to flow cytometric determination of apoptosis (left panel). The effectiveness of transfecting I κ B α -SR-cDNA/p65NF κ B-cDNA, respectively, to A549/MCF-7 cells was determined by western blot analysis (upper right panel). That I κ B α -SR overexpression in A549 cells nullified cisplatin-induced p65NF κ B nuclear translocation was confirmed by western blotting (lower right panel). ( c ) The role of IKK α / β / γ in chemoresistance was evaluated by transfecting A549 cells with dominant negative (DN) clones of IKK α / β / γ followed by cisplatin treatment and subsequent flow cytometric determination of apoptosis. The efficacy of IKK α / β / γ DN transfections (upper panel; inset) as well as expression of I κ B α and nuclear p65NF κ B in un-transfected/transfected/cisplatin-treated A549 cells (lower left panel) was determined by western blotting. Simultaneously co-immunoprecipitation experiment was employed to study the IKK γ ubiquitination (Ub-IKK γ ) in cisplatin-treated cells, and phospho-IKK β (p-IKK β ) expression was determined by western blot analysis in cisplatin-treated cells (lower right panel). ( d ) Un-transfected/p38MAPK/JNK-siRNA-transfected or SB203580/SP600125-pre-treated A549 cells were subjected to western blot analysis for determination of the levels of p38MAPK, p-p38MAPK, JNK and p-JNK in the presence or absence of cisplatin (upper left panel). In parallel, percent cell death was determined by Annexin-V/7-AAD-positivity (upper right panel). The effectiveness of pharmacological and genetic interventions of p38MAPK and JNK was demonstrated both by western blot analysis and RT-PCR (lower panel). α -Actin, histone H1 and GAPDH were used as internal controls. Values are mean±S.E.M. of five independent experiments in each case. * P

    Journal: Cell Death & Disease

    Article Title: ROS-PIASγ cross talk channelizes ATM signaling from resistance to apoptosis during chemosensitization of resistant tumors

    doi: 10.1038/cddis.2013.534

    Figure Lengend Snippet: Inhibition of NF κ B and concomitant activation of JNK during genotoxic stress are critical for inducing ‘resistance to apoptosis' switchover in drug-resistant cancer cells. ( a ) Western blotting and confocal imaging experiments were conducted to determine nuclear translocation of p65NF κ B in cisplatin-treated A549 and MCF-7 cells ( b ) Cisplatin-treated, p65NF κ B-over-expressed MCF-7 cells and I κ B α -SR-constituted A549 cells were subjected to flow cytometric determination of apoptosis (left panel). The effectiveness of transfecting I κ B α -SR-cDNA/p65NF κ B-cDNA, respectively, to A549/MCF-7 cells was determined by western blot analysis (upper right panel). That I κ B α -SR overexpression in A549 cells nullified cisplatin-induced p65NF κ B nuclear translocation was confirmed by western blotting (lower right panel). ( c ) The role of IKK α / β / γ in chemoresistance was evaluated by transfecting A549 cells with dominant negative (DN) clones of IKK α / β / γ followed by cisplatin treatment and subsequent flow cytometric determination of apoptosis. The efficacy of IKK α / β / γ DN transfections (upper panel; inset) as well as expression of I κ B α and nuclear p65NF κ B in un-transfected/transfected/cisplatin-treated A549 cells (lower left panel) was determined by western blotting. Simultaneously co-immunoprecipitation experiment was employed to study the IKK γ ubiquitination (Ub-IKK γ ) in cisplatin-treated cells, and phospho-IKK β (p-IKK β ) expression was determined by western blot analysis in cisplatin-treated cells (lower right panel). ( d ) Un-transfected/p38MAPK/JNK-siRNA-transfected or SB203580/SP600125-pre-treated A549 cells were subjected to western blot analysis for determination of the levels of p38MAPK, p-p38MAPK, JNK and p-JNK in the presence or absence of cisplatin (upper left panel). In parallel, percent cell death was determined by Annexin-V/7-AAD-positivity (upper right panel). The effectiveness of pharmacological and genetic interventions of p38MAPK and JNK was demonstrated both by western blot analysis and RT-PCR (lower panel). α -Actin, histone H1 and GAPDH were used as internal controls. Values are mean±S.E.M. of five independent experiments in each case. * P

    Article Snippet: To evaluate the involvement of p38 mitogen-activated protein kinase (p38MAPK), JNK and ATM, cells were pre-treated with a specific p38MAPK inhibitor (SB203580; 10 μ M, Calbiochem, Gibbstown, NJ, USA), JNK inhibitor (SP600125; 10 μ M, Calbiochem) 90 min before treatment and with an ATM inhibitor (Wortmanin; 20 μ M and KU55933; 10 μ M, Calbiochem) 30 min before drug treatment.

    Techniques: Inhibition, Activation Assay, Western Blot, Imaging, Translocation Assay, Flow Cytometry, Over Expression, Dominant Negative Mutation, Transfection, Expressing, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction

    SB202190 enhances cleavage of PARP when combined with glucose analogs. Western blot analysis of whole cell lysates from cells treated for 24 hours with 2-DG or D-allose alone or in combination with SB202190. Lysates were probed for cleaved and total PARP. Tubulin served as a loading control for protein. Cleaved PARP band density was quantified using ImageJ software with normalization to α-Tubulin. One representative experiment is shown, n = 3.

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: The anti-tumor efficacy of 2-deoxyglucose and D-allose are enhanced with p38 inhibition in pancreatic and ovarian cell lines

    doi: 10.1186/s13046-015-0147-4

    Figure Lengend Snippet: SB202190 enhances cleavage of PARP when combined with glucose analogs. Western blot analysis of whole cell lysates from cells treated for 24 hours with 2-DG or D-allose alone or in combination with SB202190. Lysates were probed for cleaved and total PARP. Tubulin served as a loading control for protein. Cleaved PARP band density was quantified using ImageJ software with normalization to α-Tubulin. One representative experiment is shown, n = 3.

    Article Snippet: Similarly, D-allose had anti-proliferative activity in these same cells lines when pre-treated with SB202190.

    Techniques: Western Blot, Software

    Effect of dosing regimens on the proliferation of pancreatic and ovarian cell lines following 48 hours of treatment. Cells were treated with increasing doses of 2-DG or D-allose alone or in combination with oxaliplatin (pancreatic cell lines), cisplatin (ovarian cell lines) and/or SB202190. Cell proliferation was measured using the MTT assay. The ratio of 2-DG or D-allose to the platinum analogs was 2000:1 for all doses, respectively. One representative experiment is shown, n = 6.

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: The anti-tumor efficacy of 2-deoxyglucose and D-allose are enhanced with p38 inhibition in pancreatic and ovarian cell lines

    doi: 10.1186/s13046-015-0147-4

    Figure Lengend Snippet: Effect of dosing regimens on the proliferation of pancreatic and ovarian cell lines following 48 hours of treatment. Cells were treated with increasing doses of 2-DG or D-allose alone or in combination with oxaliplatin (pancreatic cell lines), cisplatin (ovarian cell lines) and/or SB202190. Cell proliferation was measured using the MTT assay. The ratio of 2-DG or D-allose to the platinum analogs was 2000:1 for all doses, respectively. One representative experiment is shown, n = 6.

    Article Snippet: Similarly, D-allose had anti-proliferative activity in these same cells lines when pre-treated with SB202190.

    Techniques: MTT Assay

    Lactate production and HIF-1α expression in pancreatic cancer cell lines. A) . Effect of 2-DG and D-allose alone or in combination with SB202190 following 24 hours of treatment on lactate production measured in cell culture media or in cells. Values are normalized to total cell protein. One representative experiment is shown, n = 3. *p

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: The anti-tumor efficacy of 2-deoxyglucose and D-allose are enhanced with p38 inhibition in pancreatic and ovarian cell lines

    doi: 10.1186/s13046-015-0147-4

    Figure Lengend Snippet: Lactate production and HIF-1α expression in pancreatic cancer cell lines. A) . Effect of 2-DG and D-allose alone or in combination with SB202190 following 24 hours of treatment on lactate production measured in cell culture media or in cells. Values are normalized to total cell protein. One representative experiment is shown, n = 3. *p

    Article Snippet: Similarly, D-allose had anti-proliferative activity in these same cells lines when pre-treated with SB202190.

    Techniques: Expressing, Cell Culture

    HIF-1α transcriptional activity is modulated by SB202190. A) . MIA PaCa-2 cells were stably transfected with a HRE-luciferase reporter and treated for 3 24, and 48 hours with 2-DG or D-allose alone and in combination with SB202190. Luciferase activity was normalized to total protein. One representative experiment is shown, n = 3. *Indicates a p

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: The anti-tumor efficacy of 2-deoxyglucose and D-allose are enhanced with p38 inhibition in pancreatic and ovarian cell lines

    doi: 10.1186/s13046-015-0147-4

    Figure Lengend Snippet: HIF-1α transcriptional activity is modulated by SB202190. A) . MIA PaCa-2 cells were stably transfected with a HRE-luciferase reporter and treated for 3 24, and 48 hours with 2-DG or D-allose alone and in combination with SB202190. Luciferase activity was normalized to total protein. One representative experiment is shown, n = 3. *Indicates a p

    Article Snippet: Similarly, D-allose had anti-proliferative activity in these same cells lines when pre-treated with SB202190.

    Techniques: Activity Assay, Stable Transfection, Transfection, Luciferase

    Protein expression of p-ERK and p-CREB (A) or p-AKT and p-Src (B) in BxPC-3 xenografts of stress free (NS) and social stress (SS) exposed mice. Western blots (A and B) illustrating induction of p-ERK, p-CREB, p-AKT and p-Src by social stress and inhibition of these responses by celcoxib alone or celecoxib + GABA. Quantitative assessment of these changes at the protein level were determined in the ELISA assays shown in Figure C and D. Induction of all investigated phosphorylated proteins by social stress (SS) was significant ( p

    Journal: PLoS ONE

    Article Title: Celecoxib and GABA Cooperatively Prevent the Progression of Pancreatic Cancer In Vitro and in Xenograft Models of Stress-Free and Stress-Exposed Mice

    doi: 10.1371/journal.pone.0043376

    Figure Lengend Snippet: Protein expression of p-ERK and p-CREB (A) or p-AKT and p-Src (B) in BxPC-3 xenografts of stress free (NS) and social stress (SS) exposed mice. Western blots (A and B) illustrating induction of p-ERK, p-CREB, p-AKT and p-Src by social stress and inhibition of these responses by celcoxib alone or celecoxib + GABA. Quantitative assessment of these changes at the protein level were determined in the ELISA assays shown in Figure C and D. Induction of all investigated phosphorylated proteins by social stress (SS) was significant ( p

    Article Snippet: Additionally, one group each from the social stress and non-social stress populations was simultaneously treated by initial intraperitoneal injections of celecoxib (25 mg/Kg b.w., 5 days/week for 30 days) and then GABA (Sigma; 10 mg/Kg b.w., 5 days/week for 30 days) immediately thereafter on the mouse’s opposite side.

    Techniques: Expressing, Mouse Assay, Western Blot, Inhibition, Enzyme-linked Immunosorbent Assay

    Cell proliferation and migration dose response-curves. Dose response-curves for celecoxib in Panc-1 and BXPC-3 cells in vitro in the presence and absence of pretreatment for 7 days with epinephrine (15 nM). Figures A and B show cell proliferation responses in MTT assays while Figures C and D show cell migration responses. While both cellular responses were similar among the two cell lines, EC 50 values for celecoxib in Figs. B, C and D were significantly ( p

    Journal: PLoS ONE

    Article Title: Celecoxib and GABA Cooperatively Prevent the Progression of Pancreatic Cancer In Vitro and in Xenograft Models of Stress-Free and Stress-Exposed Mice

    doi: 10.1371/journal.pone.0043376

    Figure Lengend Snippet: Cell proliferation and migration dose response-curves. Dose response-curves for celecoxib in Panc-1 and BXPC-3 cells in vitro in the presence and absence of pretreatment for 7 days with epinephrine (15 nM). Figures A and B show cell proliferation responses in MTT assays while Figures C and D show cell migration responses. While both cellular responses were similar among the two cell lines, EC 50 values for celecoxib in Figs. B, C and D were significantly ( p

    Article Snippet: Additionally, one group each from the social stress and non-social stress populations was simultaneously treated by initial intraperitoneal injections of celecoxib (25 mg/Kg b.w., 5 days/week for 30 days) and then GABA (Sigma; 10 mg/Kg b.w., 5 days/week for 30 days) immediately thereafter on the mouse’s opposite side.

    Techniques: Migration, In Vitro, MTT Assay

    Mean values and SE of xenograft volumes over time from the BXPC-3 cell line in groups of mice ( n = 20). Animals were exposed to social stress (SS) or not exposed to stress (NS) in the presence and absence of celecoxib (C) or celecoxib and GABA (C+G) treatment. The xenograft volume was significantly increased ( p

    Journal: PLoS ONE

    Article Title: Celecoxib and GABA Cooperatively Prevent the Progression of Pancreatic Cancer In Vitro and in Xenograft Models of Stress-Free and Stress-Exposed Mice

    doi: 10.1371/journal.pone.0043376

    Figure Lengend Snippet: Mean values and SE of xenograft volumes over time from the BXPC-3 cell line in groups of mice ( n = 20). Animals were exposed to social stress (SS) or not exposed to stress (NS) in the presence and absence of celecoxib (C) or celecoxib and GABA (C+G) treatment. The xenograft volume was significantly increased ( p

    Article Snippet: Additionally, one group each from the social stress and non-social stress populations was simultaneously treated by initial intraperitoneal injections of celecoxib (25 mg/Kg b.w., 5 days/week for 30 days) and then GABA (Sigma; 10 mg/Kg b.w., 5 days/week for 30 days) immediately thereafter on the mouse’s opposite side.

    Techniques: Mouse Assay

    Simplified working model showing the effects of celecoxib and GABA. Xenograft growth from human pancreatic cancer cell line BxPC-3 was induced in mice by psychological stress via the interaction of β-adrenergic and COX-2 signaling pathways. Adenylyl cyclase activation downstream of the β-ARs induces the cAMP-PKA-CREB pathway, transactivates EGFR, and induces the release of EGF and AA, which in turn enhances VEGF and PGE 2 production. The inhibitory neurotransmitter GABA inhibits this signaling cascade by Gαi-mediated inhibition of adenylyl cyclase activation, whereas the reduction in PGE2 formation caused by celecoxib additionally reduced adenylyl cyclase activation downstream of the Gαs-coupled PGE2 receptors. The resulting simultaneous inhibition of VEGF, CREB, MAPK, Src, and AKT pathways contributed to the significant inhibition of tumor progression.

    Journal: PLoS ONE

    Article Title: Celecoxib and GABA Cooperatively Prevent the Progression of Pancreatic Cancer In Vitro and in Xenograft Models of Stress-Free and Stress-Exposed Mice

    doi: 10.1371/journal.pone.0043376

    Figure Lengend Snippet: Simplified working model showing the effects of celecoxib and GABA. Xenograft growth from human pancreatic cancer cell line BxPC-3 was induced in mice by psychological stress via the interaction of β-adrenergic and COX-2 signaling pathways. Adenylyl cyclase activation downstream of the β-ARs induces the cAMP-PKA-CREB pathway, transactivates EGFR, and induces the release of EGF and AA, which in turn enhances VEGF and PGE 2 production. The inhibitory neurotransmitter GABA inhibits this signaling cascade by Gαi-mediated inhibition of adenylyl cyclase activation, whereas the reduction in PGE2 formation caused by celecoxib additionally reduced adenylyl cyclase activation downstream of the Gαs-coupled PGE2 receptors. The resulting simultaneous inhibition of VEGF, CREB, MAPK, Src, and AKT pathways contributed to the significant inhibition of tumor progression.

    Article Snippet: Additionally, one group each from the social stress and non-social stress populations was simultaneously treated by initial intraperitoneal injections of celecoxib (25 mg/Kg b.w., 5 days/week for 30 days) and then GABA (Sigma; 10 mg/Kg b.w., 5 days/week for 30 days) immediately thereafter on the mouse’s opposite side.

    Techniques: Mouse Assay, Activation Assay, Inhibition

    Effect of fibulin 3 knock-down on migration of HCT116 cells. HCT116 cells with a permanent (shFib3) or transient (sh2Fib3) fibulin 3 knock-down using two different human fibulin 3 shRNAs were used. A and C , Western blot analysis of Fibulin 3 protein levels in the culture medium from HCT116 cells (non-silenced (−) and p38α knock-down (shp38α), with (shFib3 (in A ) and sh2Fib3 (in C )) or without fibulin 3 knock-down) normalized with β-actin (in cell extracts) and its quantification. p38α was used as a control of its expression. B and D , wound healing assay. Cells were pretreated with mitomycin and maintained with 2% serum, with or without 40 ng/ml of HGF (control), as indicated. Left panels , representative images from phase contrast microscope at 0 and 72 h (in B ) or at 0 and 48 h (in D ) of migration. Right panels , histograms showing the mean value ± S.E. of the percentage of wound closure at 24, 48, and 72 h (in B ) or at 48 h (in D ) with or without HGF ( n = 4), as indicated. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: p38 MAPK Down-regulates Fibulin 3 Expression through Methylation of Gene Regulatory Sequences

    doi: 10.1074/jbc.M114.582239

    Figure Lengend Snippet: Effect of fibulin 3 knock-down on migration of HCT116 cells. HCT116 cells with a permanent (shFib3) or transient (sh2Fib3) fibulin 3 knock-down using two different human fibulin 3 shRNAs were used. A and C , Western blot analysis of Fibulin 3 protein levels in the culture medium from HCT116 cells (non-silenced (−) and p38α knock-down (shp38α), with (shFib3 (in A ) and sh2Fib3 (in C )) or without fibulin 3 knock-down) normalized with β-actin (in cell extracts) and its quantification. p38α was used as a control of its expression. B and D , wound healing assay. Cells were pretreated with mitomycin and maintained with 2% serum, with or without 40 ng/ml of HGF (control), as indicated. Left panels , representative images from phase contrast microscope at 0 and 72 h (in B ) or at 0 and 48 h (in D ) of migration. Right panels , histograms showing the mean value ± S.E. of the percentage of wound closure at 24, 48, and 72 h (in B ) or at 48 h (in D ) with or without HGF ( n = 4), as indicated. *, p

    Article Snippet: Confluent cells were pre-treated with mitomycin C (25 μg/ml, Sigma-Aldrich M0503) for 30 min to inhibit cell growth.

    Techniques: Migration, Western Blot, Expressing, Wound Healing Assay, Microscopy

    Effect of MET and EGFR inhibition on heat stress and/or growth factor induced MET and EGFR signaling in HCC cells. (A) N1S1 and (B) AS30D HCC cells were treated with a dose-titration of the MET inhibitor SU11274 (0.04μM-10μM), EGFR inhibitor erlotinib (0.08μM-20μM) or vehicle control (0.1% DMSO) and assessed for viability at 72 hours using WST-1 assay. Data were normalized to vehicle control and the IC 50 estimated using non-linear regression curve fitting. Data are presented as mean±SEM of 3 independent experiments. (C) N1S1 and (D) AS30D HCC cells pre-treated with SU11274 (1μM and 10μM), erlotinib (1μM and 10μM) or vehicle control (0.1% DMSO) for 1 hour, heat stressed (45°C) or control (37°C) for 10 minutes, harvested immediately post-heat stress and whole-cell lysates were subjected to Western blotting using phospho-specific and total antibodies against MET and EGFR. β-actin was used as a loading control. (E) N1S1 cells pre-treated for 1 hour with SU11274 (5μM) or vehicle control (0.1% DMSO) followed by heat stress (45°C) or control (37°C) ± concomitant treatment with recombinant HGF (50ng/ml) or vehicle control for 10 minutes. Immediately post-heat stress whole-cell lysates were subjected to Western blotting for phospho- and total MET. β-actin was used as a loading control. (F) AS30D cells pre-treated for 1 hour with erlotinib (5μM) or vehicle control (0.1% DMSO) followed by heat stress (45°C) or control (37°C) ± concomitant treatment with recombinant EGF (50ng/ml or vehicle control for 10 minutes. Immediately post-heat stress whole-cell lysates were subjected to Western blotting for phospho- and total EGFR. β-actin was used as a loading control.

    Journal: International journal of hyperthermia : the official journal of European Society for Hyperthermic Oncology, North American Hyperthermia Group

    Article Title: Heat stress induced, ligand-independent MET and EGFR signaling in hepatocellular carcinoma.

    doi: 10.1080/02656736.2017.1385859

    Figure Lengend Snippet: Effect of MET and EGFR inhibition on heat stress and/or growth factor induced MET and EGFR signaling in HCC cells. (A) N1S1 and (B) AS30D HCC cells were treated with a dose-titration of the MET inhibitor SU11274 (0.04μM-10μM), EGFR inhibitor erlotinib (0.08μM-20μM) or vehicle control (0.1% DMSO) and assessed for viability at 72 hours using WST-1 assay. Data were normalized to vehicle control and the IC 50 estimated using non-linear regression curve fitting. Data are presented as mean±SEM of 3 independent experiments. (C) N1S1 and (D) AS30D HCC cells pre-treated with SU11274 (1μM and 10μM), erlotinib (1μM and 10μM) or vehicle control (0.1% DMSO) for 1 hour, heat stressed (45°C) or control (37°C) for 10 minutes, harvested immediately post-heat stress and whole-cell lysates were subjected to Western blotting using phospho-specific and total antibodies against MET and EGFR. β-actin was used as a loading control. (E) N1S1 cells pre-treated for 1 hour with SU11274 (5μM) or vehicle control (0.1% DMSO) followed by heat stress (45°C) or control (37°C) ± concomitant treatment with recombinant HGF (50ng/ml) or vehicle control for 10 minutes. Immediately post-heat stress whole-cell lysates were subjected to Western blotting for phospho- and total MET. β-actin was used as a loading control. (F) AS30D cells pre-treated for 1 hour with erlotinib (5μM) or vehicle control (0.1% DMSO) followed by heat stress (45°C) or control (37°C) ± concomitant treatment with recombinant EGF (50ng/ml or vehicle control for 10 minutes. Immediately post-heat stress whole-cell lysates were subjected to Western blotting for phospho- and total EGFR. β-actin was used as a loading control.

    Article Snippet: For the combination small molecule inhibitor plus heat stress experiments in , N1S1 and AS30D HCC were cells pre-treated with SU11274 (1μM and 10μM), erlotinib (1μM and 10μM) or vehicle control (0.1% DMSO) for 1 hour, heat stressed (45°C) or control (37°C) for 10 minutes, harvested immediately post-heat stress and whole-cell lysates were subjected to Western blotting.

    Techniques: Inhibition, Titration, WST-1 Assay, Western Blot, Recombinant