elexacaftor medchemexpress cat  (MedChemExpress)

Journal: Nucleic Acids Research

Article Title: ACE-tRNAs are a platform technology for suppressing nonsense mutations that cause cystic fibrosis

doi: 10.1093/nar/gkaf675

Figure Lengend Snippet: Combinatorial treatment of ACE-tRNAs and CFTR modulators significantly enhances rescued CFTR channel function. ( A ) Schematic of Fig. results demonstrating that CFTR modulators can significantly augment ACE-tRNA-dependent rescue of channel function. ( B–D ) WT and R1162X-16HBEge cells (+ hCAR) were transduced with Ad-Scr or Ad-Arg (MOI 100), pretreated with vehicle (V), lumacaftor (L, 3 μM) or elexacaftor and tezacaftor (ET, 3 μM each) for 24 h and then acutely treated with vehicle (V) or ivacaftor (I, 1 μM). (B) Relative CFTR mRNA abundance was measured in the same cell population after measurement of CFTR channel function by Ussing chamber ( n = 4–6), (C) representative I SC traces, and (D) total AUC quantification ( n = 4–7) of Ussing chamber recordings in response to sequential addition of forskolin (10 μM) and IBMX (100 μM), followed by either vehicle (V) or ivacaftor (I, 1 μM), and then by CFTR Inh -172 (20 μM). For (B) and (D), data are presented as mean ± SEM. ( E–G ) WT and W1282X cells (+hCAR) were transduced with Ad-Scr or Ad-Leu (MOI 100) and treated identically to R1162X-16HBEge cells outlined above. (E) Relative CFTR mRNA abundance quantification was done following Ussing chamber measurements of CFTR channel function ( n = 4–5), (F) representative I SC traces, and (G) total AUC quantification ( n = 4–7) from cells treated identically to cells presented in panel (D). For (B), (D), (E), and (G), data are presented as the mean ± SEM. Significance was determined by unpaired t -test or one-way ANOVA and Tukey’s post-hoc test, where ** P < 0.01, *** P < 0.001, and **** P < 0.0001 versus Ad-Scr within each genotype, ## P < 0.01 versus Ad-Arg with no L, I, or ET and #### P < 0.0001 versus Ad-Leu with no L, I, or ET.

Article Snippet: Where indicated, cells were treated with 0.3 μM SMG1i 48 h before assay and CFTR correctors 3 μM lumacaftor (L, VX-809, Selleck Chemicals, S1565) or 3 μM elexacaftor (E, VX-445, Selleck Chemicals, S8851) and 3 μM tezacaftor (T, VX-661, Selleck Chemicals, S7059) 24 h before assay.

Techniques: Transduction

Journal: Nucleic Acids Research

Article Title: ACE-tRNAs are a platform technology for suppressing nonsense mutations that cause cystic fibrosis

doi: 10.1093/nar/gkaf675

Figure Lengend Snippet: CFTR leucine variants at CF-causing PTCs are functional and responsive to modulators. ( A ) CFTR variants were cloned by site-directed mutagenesis and a stable cell line for each variant was generated in 16HBEge-G542X cells or the plasmid DNA constructs were transiently transfected into FRT cells. CFTR channel function and expression were measured for each variant (G542L shown here as an example). ( B ) Representative I SC recordings from stably integrated CFTR PTC variant 16HBEge cell lines following 24 h elexacaftor and tezacaftor treatment (ET, 3 μM each) in response to sequential addition of forskolin (10 μM) and IBMX (100 μM), followed by ivacaftor (I, 1 μM), and then by CFTR Inh -172 (20 μM). ( C ) Total AUC quantification ( n = 3–12) of Ussing chamber recordings from 16HBEge cells expressing stably integrated CFTR PTC variants in the absence (solid bars) and presence of ETI (hashed bars; n = 5–12) normalized to WT CFTR without ETI treatment. ( D and E ) Representative in-gel luminescence images of the CFTR N-terminal NLuc following SDS–PAGE electrophoresis of cell lysates in the absence (D, top) and following 24 h treatment with elexacaftor and tezacafor (ET, 3 μM each; E, top). Equal loading of cell lysates is demonstrated by CB staining of gels following in-gel luminescence imaging (D and E, bottom). Full gel images of in-gel luminescence and CB stain are presented in . ( F ) Average WT and CFTR PTC variant Band C intensity in the absence (solid bars) and presence of 24 h treatment of tezacafter and elexacaftor (ET, 3 μM each), normalized to WT CFTR Band C intensity. ( G ) Average normalized CFTR PTC variant function from (C), plotted against the average normalized Band C intensity of CFTR PTC variants from panel (F). Data from untreated (solid line) and ETI treated cells (dotted line) were subjected to linear regression analysis, with outputs of fit presented in the figure panel. ( H ) Average CFTR PTC variant activity ( I eq ) following transient expression in FRT cells with addition of ivacaftor (I, 1 μM; solid bars) or following 24 h elexacaftor and tezacaftor treatment (3 μM each) and ivacaftor treatment (1μM) during the recording (hashed bars). ( I ) WB analysis of transiently transfected CFTR PTC variants from FRT cells analyzed for CFTR PTC variant function in panel (H), with ivacaftor treatment alone (solid bars) and with elexacafter, tezacaftor and ivacafter treatment (hashed bars). WB images of all experiments in (I) are presented in . (C, F, and G) Data are presented as the mean ± SEM, n = 5–12. Significance was determined by Mann–Whitney U tests with between ETI treated and untreated cells, where * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. (H and I) Data are presented as mean ± SD of n = 2 technical replicates.

Article Snippet: Where indicated, cells were treated with 0.3 μM SMG1i 48 h before assay and CFTR correctors 3 μM lumacaftor (L, VX-809, Selleck Chemicals, S1565) or 3 μM elexacaftor (E, VX-445, Selleck Chemicals, S8851) and 3 μM tezacaftor (T, VX-661, Selleck Chemicals, S7059) 24 h before assay.

Techniques: Functional Assay, Clone Assay, Mutagenesis, Stable Transfection, Variant Assay, Generated, Plasmid Preparation, Construct, Transfection, Expressing, SDS Page, Electrophoresis, Staining, Imaging, Activity Assay, MANN-WHITNEY