Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: p38α MAPK-mediated induction and interaction of FOXO3a and p53 contribute to the inhibited-growth and induced-apoptosis of human lung adenocarcinoma cells by berberine

doi: 10.1186/1756-9966-33-36

Figure Lengend Snippet: Berberine increased the phosphorylation of p38 MAPK and ERK1/2 in a time-dependent manner. A-B , A549 cells were treated with BBR (25 μM) in the indicated times, and cell lysate was harvested and the expression of the phosphorylated or total protein of ERK1/2, p38 MAPK were measured by Western blot analysis using corresponding antibodies. GAPDH was used as loading control. The bar graphs represented the densitometry results of p-ERK (A) or p38 MAPK (B) /GAPDH as mean ± SD of at least three separate experiments. *indicates significant difference from untreated control cells (P < 0.05).

Article Snippet: Monoclonal antibodies specific for cyclinD1, p38 MAPK isoforms α, extracellular signal-regulated kinase 1/2 (ERK1/2) and their phosphor-forms were purchased from Cell Signaling Technology (Beverly, MA, USA). p38 MAPK isoforms β was ordered from AVIVA System Biology (San Diego, CA, USA).

Techniques: Phospho-proteomics, Expressing, Western Blot, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: p38α MAPK-mediated induction and interaction of FOXO3a and p53 contribute to the inhibited-growth and induced-apoptosis of human lung adenocarcinoma cells by berberine

doi: 10.1186/1756-9966-33-36

Figure Lengend Snippet: Berberine increased p53 and FOXO3a protein expression through p38α MAPK pathway. A , A549 cells were exposed to increased concentration of BBR for 24 h. Afterwards, the expression of FOXO3a and p53 protein were detected by Western blot. B - C , A549 cells were treated with SB203580 (10 μM) (B) , or p38α, β siRNAs (70 nM each) (C) for 2 h or 30 h before exposure of the cells to BBR (25 μM) for an additional 24 h. Afterwards, the expression of p38 α or β isoforms, p53 and FOXO3a protein was detected by Western blot. D , A549 cells were treated with PD98059 (20 μM) for 2 h and 30 before exposure of the cells to BBR (25 μM) for an additional 24 h. Afterwards, the expression of p53 and FOXO3a protein was detected by Western blot. The bar graphs represent the mean ± SD of p53/GAPDH and FOXO3a/GAPDH of three independent experiments. E-F , A549 cells were treated with SB203580 (10 μM) for 2 h before exposure of the cells to BBR (25 μM) for an additional 24 h. Afterwards, the cells were collected and processed for analysis of cell cycle distribution by Flow cytometry after propidium iodide (PI) staining (E) . And the percentages of the cell population in each phase (G0/G1, S and G2/M) of cell cycle were assessed by Multicycle AV DNA Analysis Software. Data are expressed as a percentage of total cells. Values are given as the mean ± SD of relative percentage of cell cycle phases from 3 independent experiments performed in triplicate. In separated experiment, the cell viability was determined using the MTT assay (F) . *indicates significant difference as compared to the untreated control group (P < 0.05). **Indicates significant difference from BBR treated alone (P < 0.05).

Article Snippet: Monoclonal antibodies specific for cyclinD1, p38 MAPK isoforms α, extracellular signal-regulated kinase 1/2 (ERK1/2) and their phosphor-forms were purchased from Cell Signaling Technology (Beverly, MA, USA). p38 MAPK isoforms β was ordered from AVIVA System Biology (San Diego, CA, USA).

Techniques: Expressing, Concentration Assay, Western Blot, Flow Cytometry, Staining, Software, MTT Assay, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: p38α MAPK-mediated induction and interaction of FOXO3a and p53 contribute to the inhibited-growth and induced-apoptosis of human lung adenocarcinoma cells by berberine

doi: 10.1186/1756-9966-33-36

Figure Lengend Snippet: Berberine increased the phosphorylation of p38 MAPK and ERK1/2 in a time-dependent manner. A-B , A549 cells were treated with BBR (25 μM) in the indicated times, and cell lysate was harvested and the expression of the phosphorylated or total protein of ERK1/2, p38 MAPK were measured by Western blot analysis using corresponding antibodies. GAPDH was used as loading control. The bar graphs represented the densitometry results of p-ERK (A) or p38 MAPK (B) /GAPDH as mean ± SD of at least three separate experiments. *indicates significant difference from untreated control cells (P < 0.05).

Article Snippet: Monoclonal antibodies specific for cyclinD1, p38 MAPK isoforms α, extracellular signal-regulated kinase 1/2 (ERK1/2) and their phosphor-forms were purchased from Cell Signaling Technology (Beverly, MA, USA). p38 MAPK isoforms β was ordered from AVIVA System Biology (San Diego, CA, USA).

Techniques: Phospho-proteomics, Expressing, Western Blot, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: p38α MAPK-mediated induction and interaction of FOXO3a and p53 contribute to the inhibited-growth and induced-apoptosis of human lung adenocarcinoma cells by berberine

doi: 10.1186/1756-9966-33-36

Figure Lengend Snippet: Berberine increased p53 and FOXO3a protein expression through p38α MAPK pathway. A , A549 cells were exposed to increased concentration of BBR for 24 h. Afterwards, the expression of FOXO3a and p53 protein were detected by Western blot. B - C , A549 cells were treated with SB203580 (10 μM) (B) , or p38α, β siRNAs (70 nM each) (C) for 2 h or 30 h before exposure of the cells to BBR (25 μM) for an additional 24 h. Afterwards, the expression of p38 α or β isoforms, p53 and FOXO3a protein was detected by Western blot. D , A549 cells were treated with PD98059 (20 μM) for 2 h and 30 before exposure of the cells to BBR (25 μM) for an additional 24 h. Afterwards, the expression of p53 and FOXO3a protein was detected by Western blot. The bar graphs represent the mean ± SD of p53/GAPDH and FOXO3a/GAPDH of three independent experiments. E-F , A549 cells were treated with SB203580 (10 μM) for 2 h before exposure of the cells to BBR (25 μM) for an additional 24 h. Afterwards, the cells were collected and processed for analysis of cell cycle distribution by Flow cytometry after propidium iodide (PI) staining (E) . And the percentages of the cell population in each phase (G0/G1, S and G2/M) of cell cycle were assessed by Multicycle AV DNA Analysis Software. Data are expressed as a percentage of total cells. Values are given as the mean ± SD of relative percentage of cell cycle phases from 3 independent experiments performed in triplicate. In separated experiment, the cell viability was determined using the MTT assay (F) . *indicates significant difference as compared to the untreated control group (P < 0.05). **Indicates significant difference from BBR treated alone (P < 0.05).

Article Snippet: Monoclonal antibodies specific for cyclinD1, p38 MAPK isoforms α, extracellular signal-regulated kinase 1/2 (ERK1/2) and their phosphor-forms were purchased from Cell Signaling Technology (Beverly, MA, USA). p38 MAPK isoforms β was ordered from AVIVA System Biology (San Diego, CA, USA).

Techniques: Expressing, Concentration Assay, Western Blot, Flow Cytometry, Staining, Software, MTT Assay, Control

Journal: Oncogene

Article Title: UVB-induced COX-2 expression requires histone H3 phosphorylation at Ser10 and Ser28

doi: 10.1038/onc.2012.71

Figure Lengend Snippet: The 14-3-3 protein isoforms might selectively recognize UVB-induced phosphorylated histone H3 and, particularly, 14-3-3ε plays an important role in UVB-induced COX-2 expression. (A) JB6 cells were either unexposed or exposed to UVB (4 kJ/m 2 ) and harvested after 1 h and total histones were prepared by acid-extraction, followed by TCA precipitation. Purified histones from JB6 cells were mixed overnight with recombinant GST-14-3-3 and immunoprecipitated, using GSH-agarose beads. After washing the beads 3 times, Western blot analysis was conducted to detect total GST and histone H3 proteins. (B) JB6 cells were exposed to UVB (4 kJ/m 2 ) and harvested after 1 h and histone proteins were acid-extracted, followed by TCA precipitation. Total histone proteins extracted from JB6 cells were mixed with recombinant GST-14-3-3 proteins and immunoprecipitated overnight, using GSH-agarose beads. After washing the beads 3 times, Western blotting was conducted to visualize the interaction between the various 14-3-3 isoforms and p-c-Jun, p-p65, p-H3S10, pH3S28, and total histone H3 (B) or Cdk9 (D). (C) JB6 cell lysates were used to examine the expression changes of the various 14-3-3 isoforms after UVB irradiation. (E) Constitutive expression of retrovirally infected HA-14-3-3ε in MCF10A cells (MCF10A-HA-14-3-3ε) was confirmed by Western blot analysis (left panel). MCF10A-HA-14-3-3ε cells were irradiated with UVB (4 kJ/m 2 ) for 1 h and disrupted with RIPA buffer, containing micrococcal nuclease. The lysates were then immunoprecipitated by HA-resin and washed with RIPA buffer three times. The interaction of HA-14-3-3ε proteins with total H3 or Cdk9 proteins was examined by Western blot (right panel). (F) After establishing JB6 cells, in which 14-3-3ε was selectively suppressed (left panel), cells were exposed to UVB (4 kJ/m 2 ) and harvested at different times (0, 1, 2, 4 h) and the abundance of COX-2 and c-Jun proteins was assessed by Western blot. (G) A schematic diagram that explains how UVB irradiation induces p38 MAPK/MSK cascade activation, leading to phosphorylation of histone H3 at Ser10 and Ser28 and subsequent recruitment of Cdk9 and RNA polymerase II phosphorylation.

Article Snippet: Micrococcal nuclease and antibodies to detect c-Jun, c-Fos, p38 MAPK, phosphorylated p38 MAPK, phosphorylated MSK1, HP1α, HP1β, HP1γ G9a, Ezh2, Suz12, and 14-3-3 isoforms (β, γ, ε, η, τ) were purchased from Cell Signaling Technology (Beverly, MA).

Techniques: Expressing, Extraction, TCA Precipitation, Purification, Recombinant, Immunoprecipitation, Western Blot, Irradiation, Infection, Activation Assay, Phospho-proteomics

Journal: Oncogene

Article Title: UVB-induced COX-2 expression requires histone H3 phosphorylation at Ser10 and Ser28

doi: 10.1038/onc.2012.71

Figure Lengend Snippet: The 14-3-3 protein isoforms might selectively recognize UVB-induced phosphorylated histone H3 and, particularly, 14-3-3ε plays an important role in UVB-induced COX-2 expression. (A) JB6 cells were either unexposed or exposed to UVB (4 kJ/m 2 ) and harvested after 1 h and total histones were prepared by acid-extraction, followed by TCA precipitation. Purified histones from JB6 cells were mixed overnight with recombinant GST-14-3-3 and immunoprecipitated, using GSH-agarose beads. After washing the beads 3 times, Western blot analysis was conducted to detect total GST and histone H3 proteins. (B) JB6 cells were exposed to UVB (4 kJ/m 2 ) and harvested after 1 h and histone proteins were acid-extracted, followed by TCA precipitation. Total histone proteins extracted from JB6 cells were mixed with recombinant GST-14-3-3 proteins and immunoprecipitated overnight, using GSH-agarose beads. After washing the beads 3 times, Western blotting was conducted to visualize the interaction between the various 14-3-3 isoforms and p-c-Jun, p-p65, p-H3S10, pH3S28, and total histone H3 (B) or Cdk9 (D). (C) JB6 cell lysates were used to examine the expression changes of the various 14-3-3 isoforms after UVB irradiation. (E) Constitutive expression of retrovirally infected HA-14-3-3ε in MCF10A cells (MCF10A-HA-14-3-3ε) was confirmed by Western blot analysis (left panel). MCF10A-HA-14-3-3ε cells were irradiated with UVB (4 kJ/m 2 ) for 1 h and disrupted with RIPA buffer, containing micrococcal nuclease. The lysates were then immunoprecipitated by HA-resin and washed with RIPA buffer three times. The interaction of HA-14-3-3ε proteins with total H3 or Cdk9 proteins was examined by Western blot (right panel). (F) After establishing JB6 cells, in which 14-3-3ε was selectively suppressed (left panel), cells were exposed to UVB (4 kJ/m 2 ) and harvested at different times (0, 1, 2, 4 h) and the abundance of COX-2 and c-Jun proteins was assessed by Western blot. (G) A schematic diagram that explains how UVB irradiation induces p38 MAPK/MSK cascade activation, leading to phosphorylation of histone H3 at Ser10 and Ser28 and subsequent recruitment of Cdk9 and RNA polymerase II phosphorylation.

Article Snippet: Micrococcal nuclease and antibodies to detect c-Jun, c-Fos, p38 MAPK, phosphorylated p38 MAPK, phosphorylated MSK1, HP1α, HP1β, HP1γ G9a, Ezh2, Suz12, and 14-3-3 isoforms (β, γ, ε, η, τ) were purchased from Cell Signaling Technology (Beverly, MA).

Techniques: Expressing, Extraction, TCA Precipitation, Purification, Recombinant, Immunoprecipitation, Western Blot, Irradiation, Infection, Activation Assay, Phospho-proteomics

Journal: Oncogene

Article Title: UVB-induced COX-2 expression requires histone H3 phosphorylation at Ser10 and Ser28

doi: 10.1038/onc.2012.71

Figure Lengend Snippet: UVB-induced COX-2 expression corresponds with activation of p38 MAPK and MSK1 in JB6 cells. (A) UVB irradiation induces COX-2 and AP-1 protein expression and activation of p38 MAPK and MSK1. (B) Pretreatment of cells with SB203580, a p38 MAPK chemical inhibitor, or H89, a MSK1 chemical inhibitor, suppresses UVB-induced COX-2 expression. (C) UVB-mediated induction of COX-2 is attenuated in JB6-DN-p38MAPK cells, compared with JB6-mock cells. (D) UVB-induced COX-2 expression is substantially suppressed in JB6-MSK1-ND or in JB6-MSK1-CD cells, compared with JB6-MSK1-WT cells (E) UVB-induced COX-2 expression is attenuated in JB6-si-MSK1 cells, compared with JB6-si-mock cells.

Article Snippet: Micrococcal nuclease and antibodies to detect c-Jun, c-Fos, p38 MAPK, phosphorylated p38 MAPK, phosphorylated MSK1, HP1α, HP1β, HP1γ G9a, Ezh2, Suz12, and 14-3-3 isoforms (β, γ, ε, η, τ) were purchased from Cell Signaling Technology (Beverly, MA).

Techniques: Expressing, Activation Assay, Irradiation

Journal: Oncogene

Article Title: UVB-induced COX-2 expression requires histone H3 phosphorylation at Ser10 and Ser28

doi: 10.1038/onc.2012.71

Figure Lengend Snippet: p38 MAPK and MSK1 are involved in UVB-induced histone H3 phosphorylation at Ser10 and Ser28 in JB6 cells. (A) Pretreatment of JB6 cells with SB203580 or H89 suppresses UVB-induced histone H3 phosphorylation at Ser10 and Ser28. JB6 cells were treated with SB203580 for 1 h prior to UVB irradiation (4 kJ/m 2 ). After UVB irradiation, cells were collected at different times (0, 1, and 2 h) and changes in histone H3 phosphorylation at Ser10 and Ser28 were measured by Western blot. (B) JB6-mock and JB6-DN-p38MAPK cells were irradiated with UVB (4 kJ/m 2 ), harvested at different times (0, 1, 2, 4 h). Changes in histone H3 phosphorylation at Ser10 and Ser28 were measured by Western blot. (C) JB6 cells were transduced with 5 different kinds of lentiviral vectors. Two independent JB6 cell clones, in which endogenous MSK1 was completely lost, were chosen for evaluating the effects of UVB-induced histone H3 phosphorylation at Ser10 and Ser28.

Article Snippet: Micrococcal nuclease and antibodies to detect c-Jun, c-Fos, p38 MAPK, phosphorylated p38 MAPK, phosphorylated MSK1, HP1α, HP1β, HP1γ G9a, Ezh2, Suz12, and 14-3-3 isoforms (β, γ, ε, η, τ) were purchased from Cell Signaling Technology (Beverly, MA).

Techniques: Phospho-proteomics, Irradiation, Western Blot, Transduction, Clone Assay

Journal: Oncogene

Article Title: UVB-induced COX-2 expression requires histone H3 phosphorylation at Ser10 and Ser28

doi: 10.1038/onc.2012.71

Figure Lengend Snippet: The 14-3-3 protein isoforms might selectively recognize UVB-induced phosphorylated histone H3 and, particularly, 14-3-3ε plays an important role in UVB-induced COX-2 expression. (A) JB6 cells were either unexposed or exposed to UVB (4 kJ/m 2 ) and harvested after 1 h and total histones were prepared by acid-extraction, followed by TCA precipitation. Purified histones from JB6 cells were mixed overnight with recombinant GST-14-3-3 and immunoprecipitated, using GSH-agarose beads. After washing the beads 3 times, Western blot analysis was conducted to detect total GST and histone H3 proteins. (B) JB6 cells were exposed to UVB (4 kJ/m 2 ) and harvested after 1 h and histone proteins were acid-extracted, followed by TCA precipitation. Total histone proteins extracted from JB6 cells were mixed with recombinant GST-14-3-3 proteins and immunoprecipitated overnight, using GSH-agarose beads. After washing the beads 3 times, Western blotting was conducted to visualize the interaction between the various 14-3-3 isoforms and p-c-Jun, p-p65, p-H3S10, pH3S28, and total histone H3 (B) or Cdk9 (D). (C) JB6 cell lysates were used to examine the expression changes of the various 14-3-3 isoforms after UVB irradiation. (E) Constitutive expression of retrovirally infected HA-14-3-3ε in MCF10A cells (MCF10A-HA-14-3-3ε) was confirmed by Western blot analysis (left panel). MCF10A-HA-14-3-3ε cells were irradiated with UVB (4 kJ/m 2 ) for 1 h and disrupted with RIPA buffer, containing micrococcal nuclease. The lysates were then immunoprecipitated by HA-resin and washed with RIPA buffer three times. The interaction of HA-14-3-3ε proteins with total H3 or Cdk9 proteins was examined by Western blot (right panel). (F) After establishing JB6 cells, in which 14-3-3ε was selectively suppressed (left panel), cells were exposed to UVB (4 kJ/m 2 ) and harvested at different times (0, 1, 2, 4 h) and the abundance of COX-2 and c-Jun proteins was assessed by Western blot. (G) A schematic diagram that explains how UVB irradiation induces p38 MAPK/MSK cascade activation, leading to phosphorylation of histone H3 at Ser10 and Ser28 and subsequent recruitment of Cdk9 and RNA polymerase II phosphorylation.

Article Snippet: Micrococcal nuclease and antibodies to detect c-Jun, c-Fos, p38 MAPK, phosphorylated p38 MAPK, phosphorylated MSK1, HP1α, HP1β, HP1γ G9a, Ezh2, Suz12, and 14-3-3 isoforms (β, γ, ε, η, τ) were purchased from Cell Signaling Technology (Beverly, MA).

Techniques: Expressing, Extraction, TCA Precipitation, Purification, Recombinant, Immunoprecipitation, Western Blot, Irradiation, Infection, Activation Assay, Phospho-proteomics

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: p38α MAPK-mediated induction and interaction of FOXO3a and p53 contribute to the inhibited-growth and induced-apoptosis of human lung adenocarcinoma cells by berberine

doi: 10.1186/1756-9966-33-36

Figure Lengend Snippet: Berberine increased the phosphorylation of p38 MAPK and ERK1/2 in a time-dependent manner. A-B , A549 cells were treated with BBR (25 μM) in the indicated times, and cell lysate was harvested and the expression of the phosphorylated or total protein of ERK1/2, p38 MAPK were measured by Western blot analysis using corresponding antibodies. GAPDH was used as loading control. The bar graphs represented the densitometry results of p-ERK (A) or p38 MAPK (B) /GAPDH as mean ± SD of at least three separate experiments. *indicates significant difference from untreated control cells (P < 0.05).

Article Snippet: Monoclonal antibodies specific for cyclinD1, p38 MAPK isoforms α, extracellular signal-regulated kinase 1/2 (ERK1/2) and their phosphor-forms were purchased from Cell Signaling Technology (Beverly, MA, USA). p38 MAPK isoforms β was ordered from AVIVA System Biology (San Diego, CA, USA).

Techniques: Phospho-proteomics, Expressing, Western Blot, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: p38α MAPK-mediated induction and interaction of FOXO3a and p53 contribute to the inhibited-growth and induced-apoptosis of human lung adenocarcinoma cells by berberine

doi: 10.1186/1756-9966-33-36

Figure Lengend Snippet: Berberine increased p53 and FOXO3a protein expression through p38α MAPK pathway. A , A549 cells were exposed to increased concentration of BBR for 24 h. Afterwards, the expression of FOXO3a and p53 protein were detected by Western blot. B - C , A549 cells were treated with SB203580 (10 μM) (B) , or p38α, β siRNAs (70 nM each) (C) for 2 h or 30 h before exposure of the cells to BBR (25 μM) for an additional 24 h. Afterwards, the expression of p38 α or β isoforms, p53 and FOXO3a protein was detected by Western blot. D , A549 cells were treated with PD98059 (20 μM) for 2 h and 30 before exposure of the cells to BBR (25 μM) for an additional 24 h. Afterwards, the expression of p53 and FOXO3a protein was detected by Western blot. The bar graphs represent the mean ± SD of p53/GAPDH and FOXO3a/GAPDH of three independent experiments. E-F , A549 cells were treated with SB203580 (10 μM) for 2 h before exposure of the cells to BBR (25 μM) for an additional 24 h. Afterwards, the cells were collected and processed for analysis of cell cycle distribution by Flow cytometry after propidium iodide (PI) staining (E) . And the percentages of the cell population in each phase (G0/G1, S and G2/M) of cell cycle were assessed by Multicycle AV DNA Analysis Software. Data are expressed as a percentage of total cells. Values are given as the mean ± SD of relative percentage of cell cycle phases from 3 independent experiments performed in triplicate. In separated experiment, the cell viability was determined using the MTT assay (F) . *indicates significant difference as compared to the untreated control group (P < 0.05). **Indicates significant difference from BBR treated alone (P < 0.05).

Article Snippet: Monoclonal antibodies specific for cyclinD1, p38 MAPK isoforms α, extracellular signal-regulated kinase 1/2 (ERK1/2) and their phosphor-forms were purchased from Cell Signaling Technology (Beverly, MA, USA). p38 MAPK isoforms β was ordered from AVIVA System Biology (San Diego, CA, USA).

Techniques: Expressing, Concentration Assay, Western Blot, Flow Cytometry, Staining, Software, MTT Assay, Control