FIGURE 5.
Influence of ePPi on the loss of phenotype induced by supernatants from chondrocytes transfected with Ank siRNA. A, effect of Ank siRNA, in the presence and the absence of 0.1 mm ePPi, on Type II collagen, and Sox-9 expression. Total RNA was extracted from chondrocytes transfected with Ank siRNA and subjected to RT-qPCR analysis. The levels of mRNA of interest were normalized to that of S29 (used as reference gene). Results are presented in histograms as means (± S.D.) over S29 value and are representative of three independent experiments. B, Western blot analysis of conditioned supernatant. Supernatants were concentrated and subjected to Western blotting. Images presented are representative of three independent experiments. C, effect of conditioned supernatant, in the presence and the absence of 0.1 mm PPi, on Tcf/Lef pathway activation. Chondrocytes were electroporated either with active (TOPFlash) or inactive (FOPFlash) Tcf/Lef reporter genes (1 μg/well of 6-well plate), and with CMV-Renilla reporter gene (200 ng/well of 6-well plate). Cells were then stimulated with conditioned supernatant from siRNA-transfected cells challenged or not with 0.1 mm ePPi for 48 h. Data are expressed as the mean luciferase activity ratio of firefly/Renilla (± S.D.) (n = 3). Statistically significant differences from supernatant produced by cells transfected with scramble are indicated as *, p < 0.05, and from cells transfected with Ank siRNA as #, p < 0.05.