Figure 1.
The best efficiently stimulation of mechanical strain was determined by MTT in MC3T3-E1 cells.
(A) The proliferation was evaluated by MTT assay under mechanical strains of 2500 µε at 0.5 Hz with different strain time (0.5 h–2.5 h) and periodicity (once or twice a day) and strain cycle (1 day–3 day). Data are represented as the mean ± SD of at least three biological replicates, *P<0.05, **P<0.01 compared with treat time (0 h) group. (B) The proliferation was evaluated by MTT assay in different intensities (1000–5000 µε) at 0.5 Hz, once a day for 1 hour over 3 consecutive days. Data are represented as the mean ± SD of at least three biological replicates, * P<0.05, ** P<0.01 compared with treat time (0 h) group.
Figure 2.
Microarray analysis of gene expression profile in MC3T3-E1 cells under mechanical strain.
(A) Hierarchical clustering analysis with the heat map under 2500 and 5000 µε at 0.5 Hz applied once a day for 1 h over 3 consecutive days in MC3T3-E1 cells. Each row represents a gene; red: up-regulated genes, Green: down-regulated genes. (B) Clustering map in gene function of MC3T3-E1 cells when stimulated by mechanical strain of 2500 µε at 0.5 Hz applied once a day for 1 h over 3 consecutive days. (C) Clustering map in gene function of MC3T3-E1 cells when stimulated by mechanical strain of 5000 µε at 0.5 Hz applied once a day for 1 h over 3 consecutive days.
Table 1.
Microarry analyses the main signaling pathways in MC3T3-E1 cells with mechanical strain.
Figure 3.
The KEGG MAPK signaling pathway showing genes differentially expressed in MC3T3-E1 cells exposed to mechanical strain.
Mechanical strains of 2500 µε, once a day at 0.5 Hz, and a periodicity of 1 h/day for 3 days. Orange frame indicates changed genes. KEGG, Kyoto Encyclopedia of Genes and Genomes.
Figure 4.
Effect of mechanical strain on the MAPK signaling pathway.
The expression values of the selected 5 genes from the MAPK signaling pathway was measured by cDNA microarray and quantitative real-time RT-PCR (qRT-PCR). MC3T3-E1 cells were stimulated under mechanical strains of 2500 µε, once a day at 0.5 Hz, and a periodicity of 1 h/day for 3 days. Data are represented as the mean ± SD of at least three biological replicates, * P<0.05, ** P<0.01.
Table 2.
The changed genes of the MAPK signaling pathway under mechanical strain of 2500 µε, at 0.5 Hz, once a day for 1 h over 3 consecutive days in MC3T3-E1 cells.
Figure 5.
Mechanical strain promotes MC3T3-E1 cells proliferation through the ERK signaling pathway.
(A–C) The protein expression of ERK and ERK-phosphorylation in different treat groups (with or without 20 µM MEK1/2 inhibitors PD98059) was detected by Western blotting with anti-ERK1/2 and anti-p-ERK1/2. GAPDH was used as an internal control. Data are represented as mean ± SD of at least three biological replicates, * P<0.05, ** P<0.01. (D) The proliferation of cells treated with or without PD98059 under mechanical strain was evaluated by MTT assay. Data are represented as the mean ± SD, of at least three biological replicates, * P<0.05, ** P<0.01.
Figure 6.
Integrin β1 and Integrin β5 have opposite effects on the phosphorylation of ERK and the proliferation in MC3T3-E1 cells.
(A) and (B) Mechanical strain induces Integrin β1 and Integrin β5 expression. The mRNA and protein expressions of Integrin β1 (A) and Integrin β5 (B) were analysised by cDNA microarray, qPCR and immunofluorescence (IF) with anti-Itgb1 and anti-Itgb5. All data are represented as mean ± SD of at least three biological replicates. * P<0.05, ** P<0.01 versus Control. (C–E) The protein expressions of ERK and ERK-phosphorylations in different treated groups (knockdown of Integrinβ1, Integrinβ5 or both simultaneously with siRNA transfection) under mechanical strain were detected by Western blotting with anti-ERK1/2 and anti-p-ERK1/2. GAPDH was used as an internal control. All data represent the mean ± SD of at least three biological replicates, * P<0.05, ** P<0.01 between the indicated groups. (F) The proliferation of cells treated with Integrin β1-siRNA, Integrin β5-siRNA or both under mechanical strain was evaluated by MTT assay. All data represent the mean ± SD of at least three biological replicates, * P<0.05, ** P<0.01 between the indicated groups.
Table 3.
Small RNA sequences.