Figure 1.
Dexamethasone dose-dependently inhibit MC3T3-E1 proliferation and induce cell death.
(A) Proliferation of MC3T3-E1 cells was measured by CCK8 (colorimetric cell counting kit-8) after cells were treated with 0, 0.001,0.01,0.1, 1.0 and 10.0 µmol/L dexamethasone for 24 hours. treatment with 1 and 10 µmol/L DEX remarkably reduced cell proliferation. B) Cell death was measured by typan blue incorporation after cells were treated with 0, 0.001,0.01,0.1, 1.0 and 10.0 µmol/L dexamethasone for 24 hours. treatment with 1 and 10 µmol/L dexamethasone remarkably increased dead cell population Values are means+SEM (n = 3). *P< 0.05 vs corresponding untreated controls.
Figure 2.
DEX-induced MC3T3-E1 apoptosis and G0/G1 arrest are abolished by RU486 pre-treatment.
(A) Assessment of apoptosis in MC3T3-E1 cells using flow cytometry with Annexin V-FITC/PI staining. The apoptotic cells to total cells ratio was significantly (P<0.05) higher in DEX group compared to the control group, RU486 group, DEX+RU group. DEX+RU group and RU486 group were implemented through 2-hour pretreatment of RU486 and then DEX/ethanol treatment. (B) The level of the cleaved-caspase 3 protein was significantly up-regulated in the DEX group, when compared to that in other groups. DEX+RU group and RU486 group (C) Cell cycle analysis using flow cytometry with PI staining, showing representive histograms of MC3T3-E1 cells in control group, DEX group, RU486 group, DEX+RU group. The distribution of the cell cycle phase was expressed as the percentage of cells in the G0–G1 phase, S phase and G2-M phase of the cell cycle. The proportion of cells in the S phase decreased markedly in the DEX group in comparison to the control group, RU486 group, DEX+RU group. The proportion of cells in the G0–G1 significantly increased in the DEX group.(P<0.05) Control group: cells treated with ethnol for 24hours. DEX group: cells treated with 1 µmol/L dexamethasone for 24 hours. RU486 group: cells pre-treated 2 hours by10µmol/L RU486 and then treated with ethnol for 24 hours. DEX+RU group: cells pre-treated 2 hours by10 µmol/L RU486 and then treated with 1 µmol/L dexamethasone for 24 hours.
Figure 3.
GRα Gene Silencing inhibited MC3T3-E1 G0–G1 arrest and apoptosis induced by DEX.
(A) Real time PCR examination of MC3T3-E1 cells in which the GRαgene function was silenced by siRNA (siGR-1, siGR-2) targeting GRαmRNA; the mRNA expression level of GRαin the siGR-1and siGR-2 groups decreased significantly (P<0.05) compared to that in the FBS group and the siC group. (B) Examination of the protein expression level of the GRαgene by Western blotting following treatment with the indicated siRNA molecules siGR-1 and siGR-2. The protein expression level of GRαsignificantly decreased in the siGR -1, and siGR -2 groups compared to that in the siC groups and FBS groups. SiGR-1 and siGR -2 group: Cells treated with siRNA molecules siGR -1 and siGR -2, respectively. siC group: Cells treated with a siRNA which had a randomized nucleotide sequence that had no significant homology to any part of the human genome. FBS group: Cells without any treatment. (C) The level of the cleaved-caspase 3 protein was significantly up-regulated in the DEX group, when compared to that in the control group, the siGR-1 group,DEX+ siGR-1 group. (D) Visualization of apoptotic cells by the TUNEL (DAB/haemotox) assay in MC3T3-E1 cells. The cell nuclei was stained with DAPI (blue) (4′,6-diamidino-2-phenylindole).And the apoptotic cells were visualized by TUNEL staining(brown). The apoptotic cells in DEX group is significantly increased compared to the control group, the siGR-1 group,DEX+siGR-1 group. (E) Assessment of apoptosis in MC3T3-E1 cell using flow cytometry with Annexin V-FITC/PI staining. Annexin V-FITC(+)PI(−) cells were considered as early apoptotic cells. The apoptotic cells to total cells ratio was significantly (P<0.05) higher in DEX group compared to the control group, the siGR-1 group,DEX+siGR-1 group. (F) Cell cycle analysis using flow cytometry with PI staining, showing representive histograms of MC3T3-E1 cells in control group, DEX group, the siGR-1 group,DEX+ siGR-1 group. The proportion of cells in the S phase decreased markedly in the DEX group in comparison to the control group, the siGR-1 group,DEX+ siGR-1 group. The proportion of cells in the G0–G1 significantly increased in the DEX group. (P<0.05) Control group: cells treated with PBS. DEX group: cells cells treated with 1 µmol/L dexamethasone. siGR-1 group: cells treated with siRNA molecules siGR -1. DEX+ siGR-1 group: cells cells treated with 1µmol/L dexamethasone plus siRNA molecules siGR -1.
Figure 4.
p53 but not granzyme A or GILZ is up-regulated by DEX treatment.
(A) Western blot analysis to describe the levels of the proteins Granzyme A, GILZ and P53 in the MC3T3-E1 cells treated with 0, 0.001,0.01,0.1, 1.0 and 10.0 µmol/L dexamethasone for 24 hours. The levels of granzyme A and GILZ did not vary significantly between the groups, and the expression of P53 was dose-dependently up-regulated. (B) Western blot analysis to describe the levels of the proteins NOXA, PUMA, p53 and p21 in the MC3T3-E1 cells. The molecular weight markers were 53 kDa (P52), 21 kDa (p21), 18 kDa (nonspecific PUMA), 24 kDa (specific PUMA), and 15 kDa (NOXA). Control group: cells treated with PBS. DEX group: cells cells treated with 1 µmol/L dexamethasone. siGR-1 group: cells treated with siRNA molecules siGR -1. DEX+siGR-1 group: cells cells treated with 1 µmol/L dexamethasone plus siRNA molecules siGR-1.
Figure 5.
p53 gene silienc by siRNA can reverse DEX induced apoptosis and cell cycle arrest of MC3T3-E1 cells.
(A) Real time PCR examination of MC3T3-E1 cells in which the p53 gene function was silenced by siRNA (sip53-1, sip53-2) targeting p53mRNA; the mRNA expression level of p53 in the sip53-1and sip53-2 groups decreased significantly (P<0.05) compared to that in the FBS group and the siC group. (B) Examination of the protein expression level of the p53αgene by Western blotting following treatment with the indicated siRNA molecules sip53-1 and sip53-2. The protein expression level of P53 significantly decreased in the sip53 -1, and sip53 -2 groups compared to that in the siC groups and FBS groups. Sip53-1 and sip53 -2 group: Cells treated with siRNA molecules sip53 -1 and sip53 -2, respectively. siC group: Cells treated with a siRNA which had a randomized nucleotide sequence that had no significant homology to any part of the human genome. FBS group: Cells without any treatment. (C) The level of the cleaved-caspase 3 protein was significantly up-regulated in the DEX group, when compared to that in the control group, the sip53 -1 group DEX+ sip53 -1 group. (D) Apoptosis as demonstarted by TUNEL (DAB/haemotox) assay in MC3T3-E1 cells. The cell nuclei was stained with DAPI (blue) (4′,6-diamidino-2-phenylindole).And the apoptotic cells were visualized by TUNEL staining(brown). The percentage of apoptotic cells in the DEX group was higher than in the control group, the sip53 -1 group DEX+ sip53 -1 group. (E) Assessment of apoptosis in MC3T3-E1 cell using flow cytometry with Annexin V-FITC/PI staining. The apoptotic cells to total cells ratio was significantly (P<0.05) higher in DEX group compared to the control group, the sip53 -1 group,DEX+ sip53 -1 group. (F) Cell cycle analysis using flow cytometry with PI staining, showing representive histograms of MC3T3-E1 cells in control group, DEX group, the sip53 -1 group,DEX+ sip53 -1 group. The proportion of cells in the S phase decreased markedly in the DEX group in comparison to the control group, the sip53 -1 group,DEX+ sip53 -1 group. The proportion of cells in the G0–G1 significantly increased in the DEX group. (P<0.05) Control group: cells treated with PBS. DEX group: cells cells treated with 1 µmol/L dexamethasone. sip53 -1 group: cells treated with siRNA molecules sip53 -1. DEX+ sip53 -1 group: cells cells treated with 1 µmol/L dexamethasone plus siRNA molecules sip53 -1.