MSCs irreversibly inhibit expression of cyclin D2. (A) C57BL/6 splenocytes (2 × 106) were stimulated with 10 μg/mL ConA in the presence or absence of MSCs (1 × 105) for 24 or 48 hours. At these times, MSCs were removed, cell pellets were lysed, and 25 mg protein was loaded onto 7% and 10% SDS-PAGE gels and was separated by electrophoresis. Proteins were transferred onto membranes and incubated with antibodies against cyclin D2 (33 kDa), p27Kip1 (27 kDa), cyclin D3 (31 kDa), cyclin E (50 kDa), cdk2 (34 kDa), cdk4 (34 kDa), cdk6 (40 kDa), and β-tubulin (55 kDa). (B) C57BL/6 splenocytes (2 × 106) were stimulated with 10 μg/mL ConA in the presence or absence of MSCs (1 × 105) for 24 and 48 hours. After 24 hours, cultures were harvested, and, after MSC removal, T cells were restimulated with ConA for another 24 hours (lane 6). Cells were then collected and analyzed for cyclin D2 expression. The stimulation of T cells in the absence of MSCs for 48 hours (lane 4) indicates the kinetics of increase in cyclin D2 expression.
