Fig. 2. Integrin α5β1 is required for LNA043-induced DKK1 secretion by C-28/I2 chondrocytes and binds LNA043.
a, LNA043-induced DKK1 secretion is integrin α5-dependent. Integrins α5 and αv were knocked down in C-28/I2 cells by siRNA (siITGA5 and siITGAV, respectively; siITGA5+V for both combined). Left, DKK1 secretion induced by LNA043 (200 μg ml−1), as analyzed by ELISA. The data represent means ± s.e.m. of 3–8 independent experiments (from left to right, ***P = 0.0003, *P = 0.0391, *P = 0.0178 and ****P ≤ 0.0001). Statistical significance was determined by one-way ANOVA with mixed-effect analysis and Šídák’s multiple comparison test. Middle, efficiency of the knockdown, as monitored by immunoblotting. GAPDH was used as a loading control. Right, quantification of the immune bands, as determined by densitometry. The data are presented as a percentage decrease ± s.e.m. versus control siRNA (siCtrl) and represent six independent experiments (****P ≤ 0.0001). Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparison test. b, LNA043 interacts with α5β1 integrin in vitro, as shown by co-IP experimentation. Recombinant human α5β1 integrin (rhα5β1) or recombinant human αvβ3 integrin (rhαvβ3) were incubated with LNA043 for 1 h at 37 °C. Immunoprecipitation was performed using a monoclonal antibody to α5β1 (anti-α5β1) or αvβ3 integrin (anti-αvβ3) followed by western blot analysis (right) with monoclonal antibodies to the α5 (left) or αv (middle) integrin subunit or ANGPTL3, which recognize LNA043 (right). One representative experiment is shown out of two to four independent experiments. c, SPR single-cycle binding studies. LNA043 (top left and top right), FN1 (bottom left) or VTN (bottom right) were immobilized on a CM5 sensor. Integrins α5β1 (left) or αvβ3 (right) served as analytes (0.6–10 µM). The sensorgrams were fitted with a 1:1 binding fit. One representative experiment is shown out of three independent experiments with comparable results. RU, resonance units.