Fig 1: SRI-41315 depletes eRF1 levels through a proteasome-mediated degradation pathway.Promoter-enhanced CFTR-G542X 16HBEge G542X cells were grown on plastic and then treated with SRI-41315 (5 µM), G418 (100 µM), or DMSO control for 48 h. a Representative western blot showing protein expression levels of the translation termination factors eRF1 and eRF3, the translation factor eIF5A, the ribosomal proteins RPL5 and RPL12, and the NMD factor proteins SMG6 and UPF2 relative to controls vinculin (A Left) or GAPDH (A Right). b Western blot quantification of eRF1 protein relative to vinculin after treatment with G418, SRI-41315, or vehicle (n = 3, data are expressed as mean ± S.D. and statistically analyzed using ordinary one-way ANOVA followed by Dunnett’s post hoc test, **p = 0.0012). c Representative western blot from cells after 20 h treatment with SRI-41315 (5 µM) alone and/or in combination with the proteasome inhibitors (S)-MG132 (10 µM) or MLN4924 (10 µM). d NanoLuc activity (normalized to total cellular protein) in 16HBEs- cells stably expressing the RT/NMD W134X or wild-type NanoLuc reporters and treated with SRI-41315 (5 µM) or MG132 (5 µM) for 24 h or MG132 (5 µM) for 12 h alone or combined. Data are expressed as the mean+/-SD. Exact p values comparing cohorts (as indicated by the brackets) were determined using two-way ANOVA Multiple Comparisons (n = 4 for the RT/NMD reporter and n = 3 for the WT reporter). This assay was independently repeated with similar results. e CFTR activity was measured in CFTR-G542X 16HBEge cells treated with MG132(10 µM), SRI-41315 (5 µM) + G418 (100 µM), and in triple combination with SRI-41315 + MG132 + G418 for 12 h. Shown are average (±S.E.M.) equivalent current (Ieq) tracings from N = 3 replicates; f the corresponding summary data calculated as area under curve (AUC) between forskolin-induced (10 µM) onset of CFTR activity and inhibition with CFTRInh-172 (20 µM) (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 < 0.0001). g Representative western blots of eRF1 and CFTR bands B and C relative to GAPDH and Na/K-ATPase in CFTR-G542X 16HBEge cells after eRF1 siRNA-mediated knockdowns alone (96 h), in combination with G418 (total 72 h with G418 replenishment at 48 h). h Corresponding quantification of westerns for eRF1 (left) and CFTR band C (right) relative to GAPDH in CFTR-G542X 16HBEge cells (n = 3, data are expressed as mean ± S.D. and statistically analyzed using ordinary one-way ANOVA followed by Dunnett’s post hoc test, eRF1 Relative to Gapdh: ***p = 0.0002 (Vehicle vs eRF1 siRNA), ***p = 0.0002 (Vehicle vs eRF1 siRNA+G418); CFTR Band C Relative to Gapdh: ****p < 0.0001 (Vehicle vs eRF1 siRNA+G418). i Representative equivalent current (Ieq) tracings corresponding to CFTR activity in CFTR-G542X 16HBEge cells after siRNA-mediated knockdown of eRF1 alone, in combination with G418, or with scramble control treatments. j Corresponding Ieq summary data calculated as area under curve (AUC) between forskolin (10 µM) and ivacaftor induced onset of CFTR activity and inhibition with CFTRInh-172 (20 µM) (n = 4, data are expressed as mean ± S.D. and statistically analyzed using ordinary one-way ANOVA followed by Tukey’s post hoc test, ****p < 0.0001). Each experiment was performed 3–4 times with n = 3–4 monolayers or wells from 96-well plates per condition on each repeat. For all panels, ***p = 0.001, ****p = 0.0001. Source data is available as a source data file.