Figure 4
This study demonstrates that COX2 inhibition is required for glafenine-mediated CFTR correction and that glafenine interacts directly with COX2. (A) Cell surface CFTR in HEK cells expressing F508del-CFTR, treated with different concentrations of glafenine (0 nM, 1 nM, 3 nM, 10 nM, 30 nM, 100 nM, 300 nM, 1 µM, 3 µM, 10 µM, and 30 μM) in combination with siRNA knockdown, of control scrambled siRNA COX1 (filled square) COX2 (open circle), or both COX1 and COX2 (open square) for 24 h before 24-h incubation with glafenine (n = 4). (B) Q-PCR to show the effectiveness of siRNA to COX1 and COX2 both when used separately and together. (C) Immunoblots showing that the reduction in mRNA resulted in less COX1 and COX2 protein, as shown in (B). (D) Graph to show the relative intensity of bands B and band C in each lane in panel C as monitored by image J. (E) Graph to show the COX enzyme activity remaining in the cells 48 h after siRNA treatment for COX-1 and COX-2 separately and together. Also the COX enzyme activity detected upon 24 h treatment with glafenine and 3 analogs compounds 1, 8 and 49 all at 10 µM. Black bars represent total COX enzymatic activity measured from cell lysates, the white and shaded bars represent COX-1 and COX-2 enzymatic activity respectively obtained due to the inhibition of recombinant COX enzymes (n = 3). Data in (B), (D) and (E) are presented as the means ± SEM.
