Figure 3
Role of progressive ankylosis gene product (ANK) in basal and hypotonically stressed eATP release from chondrocytes. For the following experiments, a hypotonic challenge was initiated by removing 35 μl of media, replacing it with 35 μl of H2O. eATP levels were measured after 10 minutes. Under control conditions, hypotonic challenge consistently increases eATP. Bars represent mean ± standard error; n = 8 for all analyses. (A) Chondrocytes were transfected with siRNA for ANK or scramble control. After 48 h, cells were exposed for 10 minutes to hypotonic (gray bars) or isotonic (black bars) media and eATP levels were measured. siANK suppressed eATP levels under basal and hypotonically challenged conditions (**P <0.01). (B, C) In parallel cultures at 48 h, mRNA and protein were isolated to assess levels of ANK mRNA (**P <0.01) and protein. Western blots compared effects on ANK versus actin. (D) Transfected chondrocytes (as described above) were exposed to media containing no additives (control) or 20 μM PPi in 100 μl of media for 30 minutes with or without hypotonic challenge. The addition of PPi did not alter eATP levels compared to controls without PPi (P >0.05). (E) Chondrocytes were treated with no additives (control), 1 mM probenecid, 5 mM probenecid or 10 mM probenecid (as described above). After the hypotonic challenge eATP levels with 1 mM probenecid were not statistically different from the control (P >0.05): 5 to 10 mM probenecid decreased eATP levels in basal and hypotonically challenged states (**P <0.01, ***P <0.001).