Fig 1.
The mechanical hypergravity device.
(A) Two horizontal rotors were made to fit the 6-well culture plates. Each rotor can also carry 2 culture flasks or 6 separated culture dishes. (B) Isometric view of the centrifuge. (C) A diagram showed the experimental setup. The control cells were placed on the top of the centrifuge and the hypergravity-treated cells were put in the rotors of the centrifuge. The centrifuge was placed in an incubator at 37°C. Thermometers were used to measure the temperature inside the centrifuge and near the control cells.
Fig 2.
Hypergravity increased the expression of OPN and Runx2.
The mRNA and protein expression levels of OPN and Runx2 in MC3T3-E1 cells after 24 h at either 1 ×g or 20 ×g are shown. (A) Comparative CT quantitation of real-time PCR results of OPN expression (mean ± SEM; n = 6). (B) Comparative CT quantitation of real-time PCR results of Runx2 expression (mean ± SEM; n = 6). (C) Immunofluorescence staining of cells under 1 ×g or 20 ×g hypergravity. Panels show individual immunostains for the OPN, Runx2, and an overlay of the two with nuclei. OPN, Runx2 and nuclei were visualized by FITC (green), TRITC (red) and DAPI (blue) (Scale bar = 25 μm). (D) The mean fluorescence intensity of OPN. ~50 cells were counted (mean ± SEM). (E) The mean fluorescence intensity of Runx2. ~50 cells were counted (mean ± SEM). (F and G) Western blotting results of OPN expression in cells and statistical analyses (n = 7). (H and I) Western blotting results of secreted OPN in medium and statistical analyses (n = 9). (J and K) Western blotting results of Runx2 expression in cells and statistical analyses (n = 6). (L and M) Cells were exposed to 1 ×g or 20 ×g for 24 h, and then incubated at 37°C. The expression of OPN was measured at 25 h, 26 h, and 27 h by western blotting. Western blotting results of OPN expression in cells and statistical analyses were shown. (n = 6) n.s. indicates no statistical difference between 1 ×g and 20 ×g. *, statistically significant difference (P < 0.05).
Fig 3.
The hypergravity-increased OPN and Runx2 expression in MC3T3-E1 cells were affected by the cell density, the preparatory adherent period and the time length for hypergravity treatment.
(A) Cells were seeded at the density of 103, 104, 105, and 106 and exposed to hypergravity. Immunofluorescence staining of cells at the density of 103, 104, and 105 under 1 ×g or 20 ×g hypergravity was shown. OPN and Runx2 were visualized by FITC (green) and TRITC (red) (Scale bar = 50 μm). (B) The mean fluorescence intensity of OPN at diverse cell densities was shown. The counted cell numbers of each class were more than 50 (mean ± SEM). (C) The mean fluorescence intensity of Runx2 at diverse cell densities was shown. The counted cell numbers of each class were more than 50 (mean ± SEM). (D, E and F) The expression of OPN and Runx2 in cells under 1 ×g or 20 ×g with the preparatory adherent period of 6 h (D), 12 h (E) and 24 h (F) were shown. (G and H) Statistical analyses of OPN (G) and Runx2 (H) expression over the diverse preparatory period (n = 6). (I and J) Western blotting results of OPN expression in cells under 1 ×g and 20 ×g for 48 h and statistical analyses (n = 4). (K and L) Western blotting results of Runx2 expression in cells under 1 ×g and 20 ×g for 48 h and statistical analyses (n = 4). (M and N) Western blotting results of OPN expression in cells under 1 ×g and 20 ×g for 72 h and statistical analyses (n = 4). (O and P) Western blotting results of Runx2 expression in cells under 1 ×g and 20 ×g for 72 h and statistical analyses (n = 4). n.s. indicates no statistical difference between 1 ×g and 20 ×g. *, statistically significant difference (P < 0.05). #, statistically significant difference between the varies cell density classes or the 6 h, 12 h, and 24 h preparatory adherent period classes (P < 0.05) (either control or hypergravity groups).
Fig 4.
Hypergravity increased the expression of OPN and Runx2 in primary osteoblasts.
(A) Immunofluorescence staining of primary osteoblasts under 1 ×g or 20 ×g for 24 h was shown. OPN, Runx2 and nuclei were visualized by FITC (green), TRITC (red) and DAPI (blue) (Scale bar = 50 μm). (B) The mean fluorescence intensity of OPN. ~50 cells were counted (mean ± SEM). (C) The mean fluorescence intensity of Runx2. ~50 cells were counted (mean ± SEM). (D and E) Western blotting results of OPN expression in primary osteoblasts under 1 ×g and 20 ×g for 24 h and statistical analyses (n = 6). (F and G) Western blotting results of Runx2 expression in cells under 1 ×g and 20 ×g for 72 h and statistical analyses (n = 6). n.s. indicates no statistical difference between 1 ×g and 20 ×g. *, statistically significant difference (P < 0.05).
Fig 5.
Hypergravity regulated OPN expression by promoting Runx2 expression.
(A) Runx2 siRNA downregulated both Runx2 and OPN expression. (B and C) Statistical analyses of results in A (n = 6). (D) Western blotting results of OPN in culture medium after Runx2 siRNA transfection. (E) Statistical analyses of results in I (n = 6). n.s. indicates no statistical difference between 1 ×g and 20 ×g. *, statistically significant difference (P < 0.05).
Fig 6.
Effects of hypergravity on the cytoskeleton.
(A) Rhodamine phalloidin (red) labeled the actin fibers and DAPI (blue) labeled the nuclei (Scale bar = 20 μm). (B) Enlarged view of actin bundles (red) (Scale bar = 25 μm). (C) The mean fluorescence intensity of the cytoskeleton. ~250 cells were counted (mean ± SEM). (D) The fluorescence distribution at the section where white lines are shown in B. (E) The mean width of actin bundles. ~200 filaments were counted (mean ± SEM). (F) The distribution of the width of actin bundles. n.s. indicates no statistical difference between 1 ×g and 20 ×g. *, statistically significant difference (P < 0.05).
Fig 7.
The effects of hypergravity on focal adhesions.
(A) FITC (green) labeled paxillin in cells, and rhodamine phalloidin (red) labeled actin filaments. Focal adhesions are indicated by arrows (Scale bar = 25 μm). (B) The FAs in cells with or without hypergravity treatment mapped to the 2D diagram coordinate of average size and intensity. (C) The mean fluorescence intensity of paxillin in focal adhesions. ~150 focal adhesions were measured (mean ± SEM). (D) The mean size of focal adhesions. ~150 focal adhesions were measured (mean ± SEM). (E and F) Western blotting results of p-FAK/FAK expression in cells and statistical analyses (n = 6). (G and H) Western blotting results of paxillin expression in cells and statistical analyses (n = 6). n.s. indicates no statistical difference between 1 ×g and 20 ×g. *, statistically significant difference (P < 0.05).
Fig 8.
Blebbistatin inhibited hypergravity-induced effects on the cytoskeleton and focal adhesions.
(A) After treated with blebbistatin, non-cortical actin alignment disappeared (Scale bar = 10 μm). (B) Blebbistatin inhibited the hypergravity-induced increase in the mean fluorescence intensity of actin fibers. ~250 cells were counted (mean ± SEM). (C) Blebbistatin treatment reduced the numbers of actin fibers in cells. ~50 cells were counted (mean ± SEM). (D) Western blotting results of p-FAK/FAK after blebbistatin treatments. (E) Statistical analyses of results in D (n = 6). n.s. indicates no statistical difference between 1 ×g and 20 ×g. *, statistically significant difference (P < 0.05). Blebbistatin (Bleb).
Fig 9.
Blebbistatin inhibited hypergravity-induced OPN and Runx2 expression.
(A and B) Western blotting results and statistical analyses of Runx2 expression in osteoblasts (n = 6). (C and D) Western blotting results and statistical analyses of OPN expression in cells (n = 6). (E and F) Western blotting results of OPN in culture medium and statistical analyses (n = 6). n.s. indicates no statistical difference between 1 ×g and 20 ×g. *, statistically significant difference (P < 0.05). Blebbistatin (Bleb).
Fig 10.
Effects of hypergravity on actin bundle intensity were inhibited by a FAK phosphorylation inhibitor.
(A and B) Western blotting results showed that PF573228 inhibited FAK phosphorylation (n = 6). (C and D) Actin bundle intensity before and after PF573228 treatment and statistical analyses. ~250 cells were counted (mean ± SEM) (Scale bar = 25 μm). n.s. indicates no statistical difference between 1 ×g and 20 ×g. *, statistically significant difference (P < 0.05).
Fig 11.
Effects of hypergravity on OPN and Runx2 expression were inhibited by a FAK phosphorylation inhibitor.
(A and B) Real-time PCR results showed the expression of OPN and Runx2 (mean ± SEM; n = 4). (C) Western blotting showed that PF573228 inhibited the hypergravity-promoted OPN and Runx2 expression. (D and E) Statistical analyses of C (n = 6). (F and G) Western blotting of OPN in the culture medium and statistical analyses (n = 5). n.s. indicates no statistical difference between 1 ×g and 20 ×g. *, statistically significant difference (P < 0.05).