Figure 4.

The viperin secretome influences chondrogenic differentiation. At day 5 in ATDC5 chondrogenic differentiation, viperin expression was either reduced by transfection of a viperin-specific siRNA duplex (Viperin siRNA) or a scrambled control siRNA duplex (Scrambled siRNA); or viperin expression was further induced by transfection of a p3xFLAG-viperin plasmid (FLAG-Viperin) or the empty p3xFLAG plasmid (FLAG-empty). The cells were then further differentiated until day 7, and viperin expression levels were determined (A). Conditioned culture supernatant (CM) from these donor cultures was harvested at day 7, and newly seeded ATDC5 cells were then differentiated with the CM supernatants for 7 days. RNA was isolated and Sox9, Col2a1, Runx2, Col10a1, and Alpl gene expression was determined in these samples (B and C). Glycosaminoglycan content (Alcian blue assay) was determined in ATDC5 cultures differentiated in CM supernatant from donor cultures in which viperin expression was reduced or further induced (D). RT-qPCR data were normalized to β-actin mRNA levels, and individual normalized values are presented as dot plots. Glycosaminoglycan content data are presented as fold change relative to the corresponding control condition. For statistical evaluation an independent samples t test was performed relative to the corresponding control condition using GraphPad Prism 5. The p values are indicated. NS, not significant. All presented data were acquired from four biological replicates, and error bars indicate means ± S.E. Graphs are representative examples of three individual experiments.