Induction of MKP-1 expression in C3H 10T1/2 cells by different stresses. (A) Time course of MKP-1 mRNA induction by arsenite (400 μM), UVC (20 J/m²), heat shock (42°C), and H₂O₂ (400 μM). MKP-1 mRNA expression was assessed by Northern blotting using total RNA. RNA loading was indicated by 18S rRNA. (B) Diagram of the MKP-1–Luc reporter. UTR, untranslated region; SV40, simian virus 40. (C) Activation of the MKP-1–Luc reporter in response to different treatments in C3H 10T1/2 cells transiently transfected with 10 μg of the luciferase reporter together with 2 μg of pCH110 (SV40-lacZ). Luciferase activity was normalized to β-galactosidase measurements and expressed in relative light units (RLU). (D) Activation of the luciferase reporter in transfected cells that stably expressed the MKP-1-Luc construct. For the luciferase assays, cells were either heat shocked at 42°C for 45 min, treated for 3 h with either 100 μM arsenite or 200 μM H₂O₂, or irradiated with UVC (10 J/m²). Data in panels C and D are means plus standard errors of the means from three independent experiments.

Role of ERK and p38 in mediating MKP-1 induction by stress in C3H 10T1/2 cells. (A) Northern blot analysis to monitor MKP-1 mRNA levels in response to arsenite (400 μM), UVC (20 J/m²), heat shock (42°C) (HS), or H₂O₂ (400 μM). U0126 (10 μM) or SB203580 (10 μM) was added to the medium 15 min before stimulation with the stress agents. Cells were harvested 1 h after exposure to stress. MKP-1 mRNA signals were normalized to 18S rRNA signals and expressed as MKP-1 mRNA induction relative to the levels in control cells. Values are means plus standard errors of the means from three independent experiments. DMSO, dimethyl sulfoxide. (B) Effects of U0126 and SB203580 on MKP-1–Luc reporter activation induced by arsenite (100 μM, 3 h). Data are means plus standard errors of the means from three independent experiments. (C) Kinetics of ERK activation. Western blotting was done with an antibody specific for phosphorylated ERK1/2 (upper panel) or an antibody recognizing total ERK (lower panel). (D) Kinetics of p38 activation. A Western blot using an antibody specific for phospho-p38 (upper panel) or an antibody for total p38 (lower panel) is shown. Cells were stimulated with arsenite (400 μM), UVC (20 J/m²), heat shock (42°C), or H₂O₂ (400 μM) for the indicated times or left untreated.

Immunofluorescent detection of Ser-10-phosphorylated histone H3. C3H 10T1/2 cells were treated with arsenite (400 μM) for the indicated times. Histone H3 phosphorylation was detected by indirect immunofluorescence. (A) Time course of histone H3 phosphorylation in response to arsenite treatment. (Upper panels) Immunofluorescent detection of phosphohistone H3 (Ser-10) (Phos-H3); (lower panels) DAPI staining of cell nuclei. Cells treated with EGF (50 ng/ml) and anisomycin (10 μg/ml) (An) for 60 min were included as a positive control. (B) Confocal microscopy of histone H3 phosphorylation. Unstimulated cells (control), cells treated with arsenite (400 μM) for 60 min, or cells stimulated with EGF (50 ng/ml) together with anisomycin (10 μg/ml) for 60 min were first stained with a rabbit phospho-histone H3 antibody (green signal) and then stained with a mouse monoclonal antibody against total histone H3 (red signal). Representative fluorescent images are shown.

Global levels of histone H3 phosphorylation-acetylation in cells stimulated with stress. C3H 10T1/2 cells were treated with arsenite (400 μM), UVC (20, 200, and 400 J/m²), heat shock (42°C), or H₂O₂ (400 μM) for the indicated times in the absence or presence of U0126 or SB203580. Cells were lysed to extract crude histone proteins. Western blot analysis was performed on the crude histone samples using an antibody specific to phospho-histone H3 (Ser-10) (Phos-H3), phosphoacetylated histone H3 (Ser-10, Lys-14) (Phos-Ac-H3), or total histone H3. (A) Histone H3 phosphorylation-acetylation in cells treated with arsenite. The graph shows the relative levels of phospho-histone H3. (B) Histone H3 phosphorylation-acetylation in cells irradiated by UVC. (C) Histone H3 phosphorylation-acetylation in cells stimulated by heat shock (42°C) (HS) or H₂O₂ (400 μM).

Activation of MSK1 in response to stress. C3H 10T1/2 cells were treated for the times indicated. MSK1 activity in cell lysates was assayed using a Crosstide peptide as a substrate. (A) Kinetics of MSK1 activation in cells stimulated with arsenite (400 μM), UVC (20 J/m²), heat shock (42°C) (HS), or H₂O₂ (400 μM). (B) Effect of U0126 or SB203580 on MSK1 activation triggered by arsenite. Cells were stimulated with arsenite (400 μM) for 60 min in the presence of either U0126 or SB203580 and harvested to assess MSK1 activity. The data are means plus standard errors of the means from three independent experiments, with each determination carried out in duplicate.

Effect of TSA on MKP-1 expression. (A) Histone H3 acetylation in cells treated with TSA. C3H 10T1/2 cells were treated with 500 ng of TSA per ml for the indicated times and harvested to extract crude histone proteins. Western blotting was performed using an antibody specifically recognizing acetyl-histone H3 (Lys-14) (Ac-H3). (B) Northern blot analysis of MKP-1 mRNA. C3H 10T1/2 cells were either pretreated with TSA (500 ng/ml) for the times indicated or received no pretreatment. Cells were either left unstimulated or further stimulated with arsenite (400 μM) for 30 min. The relative mRNA induction is quantitatively shown in the graph. Cells pretreated with TSA for 0 h refers to cells that did not receive TSA pretreatment. (C) Effect of TSA on MKP-1–Luc reporter activity in unstimulated and arsenite-stimulated C3H 10T1/2 cells. C3H 10T1/2 cells carrying the MKP-1–Luc reporter were either pretreated with 500 ng of TSA per ml for the indicated times or left untreated. Cells were then stimulated with 100 μM arsenite for 3 h. Six hours later, luciferase activity was assayed and normalized to protein amount. Data are relative to the basal luciferase activity: luciferase activity in treated cells/luciferase activity in untreated cells.

Phosphorylation-acetylation of histone H3 on the MKP-1 chromatin. (A) Phosphorylation of histone H3 (Ser-10) and association of RNA polymerase II (Pol II) with MKP-1 chromatin in control and arsenite-treated cells. C3H 10T1/2 cells were treated with 400 μM arsenite for 60 min in the presence or absence of SB203580. ChIP assays were performed using anti-phospho-H3 antibody, anti-Pol II antiserum, or preimmune serum (Ab⁻). The DNA recovered from the antibody-bound fractions as well as the DNA from input chromatin (Input) were analyzed for the presence of MKP-1, β-actin, and β-globin sequences by PCR using ³²P-labeled primers. Results of representative experiments are shown. (B) Levels of MKP-1, c-fos, β-actin, and β-globin mRNAs in cells stimulated with arsenite (400 μM) (Ars). U0126 or SB203580 (each 10 μM) was added to the medium 15 min prior to the addition of arsenite. That equal amounts of RNA were loaded was assessed by blotting with 18S rRNA. RNA from differentiated erythroleukemic BB88 cells was used as a positive control for β-globin mRNA. (C) ChIP assays performed on arsenite-stimulated cells using anti-phosphoacetyl-histone H3 antibodies. The DNA recovered from the antibody-bound fractions, the DNA from input chromatin, and the genomic DNA were analyzed by hot PCR for MKP-1 and c-fos. (D) ChIP assays performed using an antibody specific to phosphoacetylated histone H3 on cells stimulated by UVC (20 J/m², 60 min) and H₂O₂ (400 μM, 60 min). The DNA recovered was analyzed for MKP-1 and c-fos sequences by PCR. The DNA recovered was analyzed for MKP-1 and c-fos sequences by PCR. Numbers below the gels are intensities of the bands quantitated using a densitometer and expressed as the difference in band intensity in treated versus untreated cells.