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Fig. 2. Knockdown of endogenous PIASx
suppressed differentiation and matrix mineralization of MC3T3-E1 cells. (A) Effect of PIASx siRNA on the mRNA levels of osteoblast differentiation markers. Three days after confluence, osteogenic differentiation of MC3T3-E1 cells was initiated by changing the medium (day 0). At day 1 (24 hours later) cells were transfected with siRNA and RNA samples were collected at the indicated time periods. Expression was determined by semi-quantitative RT-PCR analysis. (B) Effect of PIASx siRNA on protein level. MC3T3-E1 cells were transfected with siRNA and lysates were collected after 72 hours for immunoprecipitation and western blotting as described in the Materials and Methods. (C) Effect of PIASx siRNA on the mRNA levels of osteoblast differentiation markers in calvarial osteoblasts. Three days after confluence, osteogenic differentiation of primary osteoblasts was initiated by changing the medium (day 0). At the same time, cells were transfected with siRNA and RNA samples were collected at the indicated time periods. Expression was determined by quantitative real-time RT-PCR analysis. (D) Blockage of endogenous PIASx expression by siRNA led to the inhibition of ALP activity in MC3T3-E1 cells. siRNA was transfected 24 hours after the induction of osteogenesis. 7 days later ALP staining was performed. (E) Knocking down of endogenous PIASx suppressed matrix mineralization in MC3T3-E1 cells. Cells were cultured for 27 days in DM and then subjected to Alizarin Red S (AR-S) staining. (F) To quantify the degree of mineralization, each stained culture was subjected to extraction, and samples of the AR-S extract were used for the assay. (G) A similar experiment to that described in E was performed using primary osteoblasts. Cells were cultured for 14 days in DM and then subjected to AR-S staining. (H) The degree of mineralization in primary culture was quantified.
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