Effect of CYD treatment on osteogenic differentiation. (A) MSC were left untreated (CONTROL) or treated with CYD (100 ng/ml) for 24 hours in the normal growth media and stained with phalloidin-TRITC showing less F-actin in CYD treated cells. (B) Flow cytometric analysis of MSC untreated (grey line) and treated with CYD (100 ng/ml, black line) for 24 hours stained with phalloidin-TRITC and x-axis shows the TRITC fluorescence. Grey filled is unstained control. (C) MSC were induced to undergo osteogenesis without (CONTROL) or with CYD (+CYD) for 7 or 14 days and F-actin visualised by staining with phalloidin-TRITC. (D) MSC were induced to undergo osteogenesis without (CONTROL) or with CYD (+CYD) for the indicated time period showing the fold increase in ALP positive cells. Blue colour represents alkaline phosphatase stained cells. ALP positive cells (E) or Osteocalcin expression (F) in MSC induced to undergo osteogenic differentiation without (CONTROL) or with CYD (CYD) for indicated time periods. X-axis (E,F) represents the number of days in differentiation media and y-axis indicates the fold increase in ALP positive cells compared to day 1 (CONTROL, E) or normalised OSTEOCALCIN expression (F). Representative photomicrographs or flow cytometric histogram is shown. ALP stands for alkaline phosphatase. Values are mean ± SD, n = 3-4. ** P < 0.005, *** p < 0.0005.