RhoGTPases as Key Players in Mammalian Cell Adaptation to Microgravity

Figure 4

RhoA and Rac1 activities are downregulated after 6 days of culture in simulated-microgravity conditions. Cultures were performed with C3H10T1/2 (multipotent embryonic cells) on collagen-coated microbeads (Cytodex 3, Sigma) for adipogenic induction and on Cytodex 3 beads coated with apatite minerals complexed to collagen for an osteogenic one. The adipogenic media contained 1 μM of rosiglitazone and the osteogenic media 5 mg/mL of L-ascorbic acid, β-glycerophosphate at 10⁻³ M, and retinoic acid at 10⁻⁵ M, inαMEM. Microbeads with cells were cultured for 2 days in 90 mm petri dishes (untreated for culture) with 10 mL of proliferation media (αMEM), after which the cells were switched 2 days in differentiated media, and finally left for 6 days in a NASA rotating wall vessel (RWV). In parallel, controls were realized by culturing beads in petri dishes. RhoA and Rac1 active assays were performed with specific G-LISA kits (cytoskeleton). The positive controls were pure active proteins of RhoA and Rac1 provided with the kit. The results are expressed as percentage of the positive controls; they show standard error of the mean (SEM) of samples extracted from three independent experiments and are compared with Student’s statistical -test.

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