Fig. 1. Expression of PIASx in osteoblastic cells. PIASx mRNA level in calvarial sutures (A) cultured for 6 hours in the absence (0 g) and presence (0.2 g) of TS, and in MC3T3-E1 cells (B) grown under conditions inducing differentiation toward a mature osteoblastic phenotype for 24 hours or 14 days in the absence (control) and presence (Flex) of cyclical strain by means of the FlexerCell system. (C) Expression patterns of genes encoding osteoblast differentiation markers and PIAS family proteins during osteogenic differentiation of MC3T3-E1 cells (same conditions as in B) without mechanical stimuli. Gene expression was determined by semi-quantitative RT-PCR analysis. The PCR products were analyzed by electrophoretic separation followed by Southern hybridization. These experiments were performed independently in triplicate, and representative results are shown. (D) Expression pattern of the PIASx gene during osteogenic differentiation of MC3T3-E1 cells determined by quantitative real-time RT-PCR analysis.