Fig. 2: CFTR expression and function is enhanced after combination treatment with SRI-37240 and aminoglycosides in FRT cells stably transduced with human CFTR-G542X cDNA.
From: A small molecule that induces translational readthrough of CFTR nonsense mutations by eRF1 depletion
FRT monolayers were treated with SRI-37240 (10 µM unless indicated otherwise)+/−G418 (100 µg/mL) for 48 h on a liquid–liquid interface. a Forskolin-induced CFTR conductance after treatment with SRI-37240 at 1, 3, 10, and 30 µM concentrations alone (blue squares) and in combination with G418 (purple squares) (n = 4, data are statistically analyzed using Two-way ANOVA followed by Sidak’s post hoc test, p = 0.0006 (SRI-37240 vs SRI-37240 (10 uM) + G418), p < 0.0001 (SRI-37240 vs SRI-37240 (30 uM) + G418). b Representative conductance tracings after treatment with SRI-37240+/−G418. c Corresponding summary of conductance measurements of SRI-37240+/−G418 after forskolin stimulation (represented by corresponding colors as in raw tracings in Fig. 2b) and ivacaftor (iva) (light gray) induced CFTR conductance and CFTRInh-172 (dark gray) mediated inhibition; also expressed as % of wild-type CFTR conductance in isogenic FRT cells (i.e., 25 mS/cm2) (n = 3, data are expressed as mean ± S.D. and statistically analyzed using repeated measures one-way ANOVA followed by Tukey’s post hoc test). ****p < 0.0001 (G418 vs SRI-37240 + G418), ****p < 0.0001 (SRI-37240 vs SRI-37240+/−G418). d Representative western blot showing increased CFTR band C (fully mature) and band B (core glycosylated) after combination treatment with SRI-37240+/−G418. Total protein loaded: WT = 10 µg and G542X = 30 µg). e Densitometric quantification of full-length CFTR Band C (mature form) relative to α-tubulin from FRT cells expressing wild-type or G542X CFTR cDNAs after treatments with SRI-37240 and G418 (alone or in combination). f Steady-state CFTR transcript levels by real-time RT-PCR relative to GAPDH after treatment with SRI-37240+/−G418. Data is the representation of n = 3 monolayers per condition and 2–4 experimental repeats for each experiment (n = 3, data are expressed as mean ± S.D. and statistically analyzed using ordinary one-way ANOVA followed by Tukey’s post hoc test, p = 0.0019 (Vehicle vs G418), p = 0.0014 (Vehicle vs SRI-37240), p < 0.0001 (Vehicle vs SRI-37240 + G418), p = 0.002 (SRI-37240 vs SRI-37240 + G418). For all panels, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Source data is available as a source data file.
