Fig 1.
Hypergravity down-regulated c-fos expression in ATDC5 cells.
(A) mRNA expression of c-fos and c-jun in ATDC5 cells after loading 32G of hypergravity 1 hour daily for 3 days. (B) Time-course of c-Fos mRNA expression after loading 32G of hypergravity for 0, 30, 60, and 120 min. (C) Intensity-dependent effect of hypergravity on c-Fos mRNA expression in ATDC5 cells after loading 1G, 18.7G, 33.3G, 52.0G, 207.9G, and 467.9G for 60 min. mRNA expression of all the genes were analyzed by real-time PCR and normalized by Gapdh and Rpl13a expression. The bars represent the mean ± standard error. The results are normalized to the control sample (n = 4). p values were obtained by performing a Student’s test; *p<0.05, **p<0.005 compared with the control sample.
Fig 2.
Involvement of actin cytoskeleton in the down-regulation of c-fos by hypergavity.
Staining of cell nuclei (with DAPI, in blue) and actin filaments (with rhodamin phalloidin, in red) after loading 18.7G of hypergravity for 60 min; (A) 1G (B) 18.7G. The scale bar represents 50μm.(C) Actin threshold area normalized to the number of nucleus was quantified from the result of Actin immunostaining (n = 4). The results were expressed as relative amounts against the expression of control sample (n = 4). Inhibitor tests: (D) Y27632 and GSK429286 and (E) Cytochalasin D and Jasplakinolide were utilized respectively as a ROCK inhibitor and an actin polymerization inhibitor. c-fos mRNA expression was analyzed by real-time PCR and normalized to Gapdh and Rpl13a expression. Each value is the average of three experiments. The bars represent the mean ± standard error. p values were obtained by performing a Student’s test; *p<0.05, **p<0.005 compared with the control sample.
Fig 3.
Involvement of PI3K signaling pathway in the down-regulation of c-fos by hypergravity.
Wortmanin Inhibitor tests in ATDC5 cells under hypergravity for 60 min. (A) c-fos mRNA expression was analyzed by real-time PCR and normalized to Gapdh and Rpl13a expression. Wortmanin was utilized as a PI3K inhibitor. (B) Western blotting was carried out to check the change in phosphorylated-Akt (p-Akt), total-Akt, and α-tubulin protein levels after loading 18.7G of hypergravity for 60 min. Wortmanin was added as a PI3K inhibitor to confirm the involvement of the PI3K pathway, particularly Akt. (C) P-Akt levels were normalized to total Akt protein levels. Each value is the average of three experiments. The bars represent the mean ± standard error (n = 3). p values were obtained by performing a Student’s test; *p<0.05, **p<0.005 compared with the control sample.
Fig 4.
Schematic diagram of signaling pathways involved in c-fos expression in cells under hypergravity.