The proportion of cells in the G2/M phase was decreased significantly in the simulated microgravity + pcDNA3.1\cyclin B1 group compared with that in the simulated microgravity + pcDNA3.1 group, but was similar to the proportion of cells in G2/M in the control + pcDNA3.1 group (Physique ?(Physique5B5B and C). simulated microgravity was mainly because of decreased cyclin B1 expression. Furthermore, miR\181c\5p directly inhibited cyclin B1 protein translation by binding to a target site in the 3UTR. Lastly, we exhibited that inhibition of miR\181c\5p partially counteracted cell cycle arrest and decreased the osteoblast proliferation induced by simulated microgravity. In conclusion, our study demonstrates that simulated microgravity inhibits cell proliferation and induces cell cycle arrest in the G2 phase in primary mouse osteoblasts partially through the miR\181c\5p/cyclin B1 pathway. This work may provide a novel mechanism of microgravity\induced detrimental effects on osteoblasts and offer a new avenue to further investigate bone loss induced by mechanical unloading. assessments or one\way analysis of variance was used to compare the CL 316243 disodium salt means. The test was considered to be significant when test was performed for each sample against control samples. * em P /em ? ?0.05 and ** em P /em ? ?0.01, when compared with the stationary control. 3.2. Simulated microgravity induces osteoblast cell cycle arrest in the G2 phase We performed FCM assays to evaluate the effects of simulated microgravity on cell cycle distribution in primary mouse osteoblasts. The proportion of cells in the G2/M phase was increased significantly, while the proportion of cells in the G0/G1 and S phases was decreased in the simulated microgravity group compared with that in the control group (Physique ?(Physique2A2A and B). To further clarify the exact ratio of cells in the M phase, we performed immunofluorescence assays for the expression of histone H3 (phospho Ser10). Physique ?Physique2C2C and D illustrated that this mitotic index of osteoblasts was decreased in the simulated microgravity group and was significantly increased in cells pretreated with the mitotic inhibitor nocodazole (which is known to block cell cycle progression in the M phase through disruption of mitotic spindles, and which served as a positive control). Moreover, the expression of histone H3 (phospho Ser10) was diminished in the simulated microgravity group and was noticeably increased in the nocodazole group compared with the control group (Physique ?(Figure22E). Open in a separate window Physique 2 Cell cycle of osteoblasts is usually Rabbit polyclonal to ALP arrested in the G2 phase (as opposed to the M phase) in response to simulated microgravity. A and B, Flow cytometry analysis of primary mouse osteoblasts CL 316243 disodium salt treated with simulated microgravity was performed to test the cell cycle distribution. A, Representative histograms indicate the cell cycle distribution in different groups. The relative DNA contents of cells were determined by PI staining. B, The percentage of cells in each cycle stage was quantified (n?=?5). C\E, The effect of simulated microgravity around the mitosis index of osteoblasts was detected by immunofluorescence for histone H3 (phospho Ser10). C, Cells were seeded onto glass coverslips and, after simulated microgravity treatment for 48?h, cells were fixed, permeabilized and subjected to staining with Hoechst (blue) to visualize nuclei and with anti\histone H3 (phospho Ser10) primary antibody and Alexa Fluor 488 conjugated secondary antibody (green) to visualize cells undergoing mitosis. Images were analysed using a confocal microscope. D, Histogram of the percentage of histone H3 (phospho Ser10)\positive cells from these groups. The mitotic index was expressed as the ratio of histone H3 (phospho Ser10)\positive cells to total Hoechst positive cells (n?=?3). E, Western blot analysis of histone H3 (phospho Ser10) expression was decided in cell lysates from primary mouse osteoblasts. The total protein loaded per lane was 40?g. Detection of GAPDH on the same blots was used to verify equal loading among the various lanes (upper). Histogram of the relative expression of histone H3 (phospho Ser10) present in cells from each group quantified by camera\based detection of emitted chemiluminescence (lower) (n?=?4). Cells treated with 0.5?g/mL nocodazole (a mitotic inhibitor) for 24?h were used as a positive control. The results were expressed as the mean??SD with a one\way ANOVA with a SNK\q test. * em P /em ? ?0.05 and ** em P /em ? ?0.01, compared with the stationary control. 3.3. Simulated microgravity has no effects around the cellular localization, expression and activity of Cdc2 kinase In the eukaryotic cell cycle, activation of Cdc2 kinase is required for cells to enter mitosis. We asked whether CL 316243 disodium salt the simulated microgravity\induced G2 arrest in primary mouse osteoblasts was because of the inactivation of the cyclin B1/Cdc2 kinase complex. As this complex is maintained in an inactive form through phosphorylation of the Cdc2 residues Thr14 and Tyr15, we performed an immunostaining assay to study the cellular localization and expression of Cdc2 and Cdc2 (phospho Tyr15) in osteoblasts under simulated microgravity conditions. As shown in Physique ?Physique3A,3A, Cdc2 expression in the control and simulated microgravity groups was localized intracellularly, but was not nuclear. In contrast, Cdc2 had translocated into the nucleus in nocodazole\stimulated cells (Physique ?(Figure3A).3A). Interestingly, there was no difference in the cellular localization and fluorescence intensity of Cdc2 in the control and simulated.