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. Author manuscript; available in PMC: 2010 Sep 11.
Published in final edited form as: Circ Res. 2009 Aug 6;105(6):575–584. doi: 10.1161/CIRCRESAHA.109.202333

Figure 6. HSP70 mediates the formation of condensations, ALP and mineralization induced by IL-6 in CVC.

Figure 6

(A) CVC were treated with IL-6 (10 or 50 ng/ml), and HSP70 in the media was determined by immunoblotting after 24 hours.

(B-E) CVC were treated with IL-6 (50 ng/ml) alone or with anti-HSP70 antibodies or control IgG (300 ng/ml). After 12 hours, activation of SMAD1/5/8 was determined by pSMAD1/5/8 immunoblotting and compared to pSMAD2/3 and total SMAD (B). After 2 days, formation of condensations was examined (C), and ALP activity was determined (D). After 8 days, accumulation of calcium was determined (E).

(F-H) CVC were transfected with siRNA to HSP70 or scrambled control IgG. The decrease in HSP70 expression was confirmed by real-time PCR and immunoblotting after 24 hours (E). The CVC were treated with IL-6 (50 ng/ml) for up to 8 days. ALP activity and calcium accumulation were determined after 2 and 8 days, respectively (F, G).

Asterisks indicate statistically significant differences compared to control (no treatment). *, p<0.05; ***, p < 0.001; Tukey’s test.

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