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. 2012 Jun 7;287(31):25706–25714. doi: 10.1074/jbc.M112.361360

FIGURE 5.

FIGURE 5.

Knockdown of Klf4 attenuated high phosphate-induced phenotypic switching of SMCs. A, cultured SMCs were transfected with siRNA specific for Klf4 (siKlf4) or a scramble sequence (siScr) and incubated with normal (NP) or high (HP) phosphate concentration for 2 days. Expression of Klf4 and SRF in the nuclear fraction of SMCs was examined by Western blotting (n = 3). B, SMCs were transfected with siRNA for Runx2 (siRunx2) or siScr, followed by transfection of FLAG-Runx2 expression plasmid or control plasmid (Ct). Expression of Runx2 (anti-FLAG antibody) and GAPDH was examined by Western blotting (n = 3). C–G, cultured SMCs were transfected with siKlf4, siRunx2, and/or siScr and incubated with normal (NP) or high (HP) phosphate concentration for 6 days. Expression of SM α-actin (C), Runx2 (D), osteopontin (E), and ALP (F) mRNA was determined by real-time RT-PCR. Calcium deposition was measured and normalized by cellular protein content (G). Values represent the means ± S.E. *, p < 0.05 compared with SMCs with normal phosphate medium (n = 3).