Fig. 2.

Molecular chaperone activity maintains the limited transport competence of ΔF508- and P67L-CFTR. a FPMA of WT and ΔF508-CFTR calculated from PM density and short-circuit current (I _sc) with and without 24 h 3 μM VX-809 rescue, expressed as percentage of WT-CFTR expressing CFBE (left panel). I _sc was measured after sequential addition of 20 μM forskolin (fsk) and 100 μM genistein (gen), followed by CFTR inhibition with Inh₁₇₂ (172, 20 μM). The FPMA (right panel) was calculated as the ratio of PM density and I _sc, and normalized to WT. Means ± SEM, n = 3; error bars in the right panel are error propagation. b The effect of chaperone or co-chaperone knockdown on PM density, function and FPMA of the ΔF508-3HA in CFBE. The rescued ΔF508-CFTR (30 °C, 48 h) was unfolded for 2 h at 37 °C. Hsp90α (90α), Hsp90β (90β), DNAJA1 (JA1), DNAJA2 (JA2), Aha1, Hsp70, and Hsc70 were silenced with 50 nM siRNA. Non-targeted siRNA served as controls. Function was determined by YFP quenching assay. c The effect of Hsc70 and Hsp90 inhibition on the PM density and function of the ΔF508- and WT-CFTR-3HA in CFBE. The rescued ΔF508-CFTR (30 °C, 48 h) was unfolded (2 h at 37 °C) in the presence of 5 μM Pif and/or 5 μg ml⁻¹ GA. I _sc was measured as in a. Representative measurements are in the top panels. d The effect of Hsc70 and Hsp90 activity on the FPMA of the ΔF508- and P67L-CFTR in CFBE cells. Experiments were performed as in c. e I _sc and PM density of P67L-CFTR in CFBE as described in c. f The effect of Hsc70 and Hsp90 activity on ΔF508-CFTR FPMA in CFBE cells. CFBE cells, expressing ΔF508-CFTR with second site mutations or rescued ΔF508-CFTR (30 °C, 48 h), were treated for 2 h at 37 °C with 5 μM Pif and 5 μg ml⁻¹ GA. Experiments were performed as in c. g Conformational rescue of the ΔF508-CFTR by low temperature plus VX-809 (3 μM, 24 h) prevents the loss of ΔF508-CFTR FPMA by Pif + GA in CFBE cells. Experiments were performed as in c. Data are means ± SEM, n = 3–4. *P < 0.05, **P < 0.01 and ***P < 0.001