Surface Immunostaining Profiles of CF Variant Libraries.

Flow cytometry was used to characterize the distribution of CFTR surface immunostaining intensities among recombinant CANX KO and parental HEK293T cells expressing a pool of 234 CF variants. A) A histogram depicts the distribution of CFTR surface immunostaining intensities among recombinant CANX KO (orange) and HEK293T (blue) cells treated with vehicle (DMSO). The mean fluorescence intensities of parental HEK293T cells stabling expressing WT or ΔF508 CFTR are shown for reference. B) A histogram depicts the distribution of CFTR surface immunostaining intensities among recombinant parental HEK293T cells expressing the CF variant library treated with vehicle (light blue) or with 3 µM VX-661 + 3 µM VX-445 (dark blue). The mean fluorescence intensities of parental HEK293T cells stabling expressing WT or ΔF508 CFTR are shown for reference. C) A histogram depicts the distribution of CFTR surface immunostaining intensities among recombinant CANX KO cells expressing the CF variant library treated with vehicle (orange) or with 3 µM VX-661 + 3 µM VX-445 (red). The mean fluorescence intensities of CANX KO cells stabling expressing WT or ΔF508 CFTR are shown for reference.

Influence of Calnexin on CF Variant Plasma Membrane Expression.

The difference plasma membrane expression (PME) in CANX KO cells relative to the parental HEK293T cells is shown for 234 CF variants. A) The change in expression for each variant is plotted against its position within the CFTR sequence. The boundaries of the six CFTR domains highlighted for reference. B) A box and whisker plot depicts the statistical distributions for the change in variant PME across variants within each subdomain. The upper and lower edges of the box reflect the 75th and 25th percentile values and the upper and lower whiskers reflect the 90th and 10th percentile values, respectively. The lines within each box represent the median value and the squares within the box represent the average. ** denotes p < 0.01 for a Mann-Whitney U-test. C) Values for the change in PME for each variant are projected onto their corresponding residues within a structural model of CFTR (5UAK).

Influence of Calnexin on the Sensitivity of CF Variants to VX-445.

Deep mutational scanning was used to quantitatively compare the effect of 3 μM VX-445 on the plasma membrane expression (PME) of 234 CF variants in CANX KO cells relative to the corresponding parental HEK293T cell line. A) A scatter plot depicts the change in the VX-445 response in CANX KO cells relative to parental cells against the corresponding PME in parental HEK293T cells for each CF variant. B) A scatter plot depicts the change in the VX-445 response in CANX KO cells relative to parental cells against the position of the mutated residue for each CF variant. C) The change in the VX-445 response in CANX KO cells relative to parental cells for each CF mutant is projected on to the structure of VX-445 bound ΔF508 CFTR (PDB 8EIG). The structure of VX-445 is shown in yellow for reference. Negative values indicate a weaker response and positive values indicate a greater response to VX-445. Transmembrane helices 10 and 11 and ICL4 are depicted in green.

Influence of Calnexin on the Sensitivity of CF Variants to VX-445+ VX-661.

Deep mutational scanning was used to quantitatively compare the effect of 3 μM VX-445 + 3 μM VX-661 on the plasma membrane expression (PME) of 234 CF variants in CANX KO cells relative to the corresponding parental HEK293T cell line. A) A scatter plot depicts the change in the VX-445 + VX-661 response in CANX KO cells relative to parental cells against the corresponding PME in parental HEK293T cells for each CF variant. B) A scatter plot depicts the change in the VX-445 + VX-661 response in CANX KO cells relative to parental cells against the position of the mutated residue for each CF variant. C) The change in the VX-445 + VX-661 response in CANX KO cells relative to parental cells for each CF mutant is projected on to the WT CFTR structure (PDB 5UAK). Negative values indicate a weaker response and positive values indicate a greater response to VX-445 + VX-661.

Interactome of CF Variants in CANX KO Cells.

Proteomic mass spectrometry was used to compare the protein-protein interactions formed by CF variants in the context of CANX KO cells relative to the parental cell line. A) A cartoon depicts the workflow for multiplexed affinity purification-mass spectrometry (AP-MS) experiments. B) Violin plots depict the log2 fold change in protein abundances of various classes of interactors that associate with CF variants in CANX KO cells relative to their corresponding abundance in the parental cell line. Each data point represents individual protein interactors that are grouped according to their pathways. C-E) Violin plots depict the log2 fold change in protein abundances of various classes of interactors that associate with ΔF508 in CANX KO cells treated with various correctors relative to their corresponding abundance in the parental cell line under identical treatment conditions. Each data point represents individual protein interactors that are grouped according to their pathways. Statistical significance is denoted as follows: Red asterisks indicate a significant deviation from zero, assessed using a one-sample t-test and Wilcoxon test against a hypothetical value of 0. Black asterisks denote significant differences from wild-type (WT) levels, determined using a repeated measures one-way ANOVA with Geisser-Greenhouse correction (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

Influence of Calnexin on the Functional Rescue of CF Variants.

The functional properties of CF variants are compared in various cells that feature endogenous CANX expression or deficient CANX expression under various experimental conditions. A) Flow cytometry was used to monitor CFTR-mediated quenching of a halide sensitive YFP (hYFP) relative to an mKate expression marker upon activation of stably expressed CFTR variants with 25 µM forskolin (Fsk) and 50 µM genistein in parental HEK293T and CANX KO HEK293T cells. Representative flow cytometry measurements of single-cell hYFP: mKate ratios are plotted over time following CFTR activation for recombinant cells stably expressing a series of CF variants. Fitted curves for single exponential fit decay are shown for reference. B-C) Bar graphs depict the fitted half-lives of the hYFP quenching reactions among cells expressing each indicated CF variant in parental HEK293T cells (CANX+, open bars) and CANX KO cells (CANX-, dashed bars) treated with B) DMSO or with C) 3 µM VX-661, 3µM VX-445, and 10 µM VX-770. Values represent the mean ± SEM (n = 3). D-E) FRT monolayers transiently expressing D) ΔF508 or E) WT CFTR were cultured 5 days on permeable transwells and chronically treated with CFTR modulators (72hr, 5µM), vehicle control (DMSO), and/or various siRNA (72hr, 100nM). Bar plots depict average short-circuit currents (ΔISC) following acute application of 10µM Fsk, 5µM VX-770, and/or 10µM of the CFTR-specific inhibitor-172 (Inh172). F) A bar graph depicts the total functional activation of ΔF508 CFTR that is achieved under various conditions upon stimulation with Fsk and VX-770 relative to WT stimulated with Fsk. Values represent mean ± SEM (n = 4). Asterisks annotate statistical evaluation of Fsk ± VX-770 values compared to non-specific (NS, red) or CANX-specific (blue) siRNA.*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 (two-way ANOVA). Currents from non-treated cells (NT) are shown for reference.