Figure 3.

(See previous page). Effects of the combination treatment with cysteamine and epigallocatechin gallate (EGCG) on human airway epithelial cells and mouse lungs. (A) CFBE41o-cells were transfected with F508del-CFTR at 37 °C. After transfection, the cells were incubated for 18 h with or without cysteamine (250 μM) and then kept in medium for 24 h or 48 h in the presence or absence of EGCG (80 μM), with or without CFTRinh-₁₇₂ (20 μM). Cycloheximide (CHX) (100 μg ml⁻¹) was added during the last 8 h of incubation. The lack of CHX toxicity in this model is reported in Fig. S3A. Top, surface biotinylation followed by purification of streptavidin-bound PM proteins and immunoblot with anti-CFTR (clone CF3, Abcam). FLOT1 (clone C-2 Santa Cruz Biotechnology) confirmed cell surface protein-specific localization. Bottom, densitometric measurement of the residual CFTR at the PM expressed as fold increase of the initial amount (medium) normalized to FLOT1 levels, Mean ± SD of triplicates of independent experiments; °°P < 0.01 compared to medium (ANOVA). (B) Effects of EGCG (top) and cysteamine (bottom) on the activity of either the CSNK2A/α subunit or the CSNK2A₂-CSNK2B₂ (α₂β₂) holoenzyme acting on the synthetic peptide substrate RRRADDSDDDDD. IC₅₀ values represent the mean of 3 independent experiments with the SD not exceeding 10%. (C) CFBE41o-cells were transfected with F508del-CFTR and incubated with cysteamine and then kept up to 48 h in medium alone as in (A). During cysteamine washout, the cells were incubated with medium or medium added with the CSNK2 inhibitor CX-4945 (5 μM) in the presence or absence of CFTRinh-₁₇₂ (20 μM). CHX was added to the system as in (A). Top, surface biotinylation followed by purification of streptavidin-bound PM proteins and immunoblot with anti-CFTR (clone CF3, Abcam). FLOT1 (clone C-2 Santa Cruz Biotechnology) confirmed cell surface protein-specific localization. Bottom, densitometric measurement of the residual CFTR at the PM expressed as fold increase of the initial amount (medium) normalized to FLOT1 levels. Mean ± SD of triplicates of independent experiments, °P < 0.05, °°P < 0.01 compared to medium (ANOVA). (D) Tnf (top) and Cxcl2 (bottom) transcription levels in lung homogenates from 10-12 wk-old Cftr^F508del mice either immediately after treatment with vehicle or cysteamine for 7 d or alternatively after a latency of 10, 20, or 30 d without cysteamine treatment. During this “washout” period, the mice were either left untreated or treated with cysteamine. Mean ± SD of triplicates of 5 mice per group, °P < 0.05, °°°P < 0.001 compared to vehicle and **P < 0.01, ***P < 0.001 compared to 30 d of washout without cysteamine (ANOVA).