Figure 7

Translocation of NFAT3 and SMAD3 to the nucleus. Translocation of A) NFAT3 and B) SMAD3 in the nuclei of human osteoarthritic chondrocytes (n = 7 to 10). For NFAT3, cells received no treatment (control) or were treated with TGF-β for 30 minutes or with ionomycin for 90 minutes without or with TGF-β added for the last 30 minutes. For SMAD3, cells received no treatment (control) or were treated with ionomycin for 60 minutes or with TGF-β for 90 minutes without or with ionomycin added for the last 60 minutes. Representative immunocytochemistry of the translocation of A) NFAT3 in the control, TGF-β, ionomycin, and ionomycin/TGF-β treated osteoarthritic chondrocytes for the abovementioned time period, and B) SMAD3 in the control, ionomycin, TGF-β, and TGF-β/ionomycin-treated cells for the abovementioned time period. The percentage of NFAT3 and SMAD3 (green stain) nuclear translocation was calculated relative to the number of total cells (nuclei stained blue with DAPI). The white arrow-heads identify positive-nuclei. P values were assessed by the Mann–Whitney test comparing treatments to controls or as underlined. Effect of C) TGF-β on the ionomycin-induced expression of miR-140 and of D) ionomycin on the TGF-β-induced expression of miR-140. Human osteoarthritic chondrocytes (n = 7) were treated for 8 hours with ionomycin with or without the addition of TGF-β for the last 6 1/2 hours, or with TGF-β for 8 hours without or with the addition of ionomycin for the last 6 1/2 hours. RNA was extracted and the expression of miR-140 was measured. C) and D) each control (ionomycin and TGF- β, respectively) was assigned an arbitrary value of 1 and the effect of the double treatment evaluated as fold change over control. P values were assessed by the one-sample t-test, comparing the double treated chondrocytes to the autologous control. DAPI, 4’,6-diamidino-2-phenylindole, dihydrochloride; TGF-β, transforming growth factor β.