Silencing of HSF1 improves F508del folding and its cell surface stability.

Abstract

<p>(A) Total protein synthesis on S³⁵ labeled samples from WT- or F508del-CFTR expressing cells treated with control or siHSF1. Results are shown as percent of WT for total protein normalized by number of cells in each condition (mean ± SEM, <i>n</i> = 3; * indicates <i>p</i><0.05 relative to WT). (B) Pulse-chase of F508del-CFTR in response to siHSF1 treatment. Numbers show percent of F508del relative to time 0 (T = 0; <i>n</i> = 2). (C) F508del-CFTR immunoblots and quantification in control or siHSF1 transfected cells before (T = 0) and after cycloheximide (CHX; 50 µM) chase for the indicated time (h). Results are shown as percent of band-B or band-C at T = 0 (mean ± SD, <i>n</i>≥4). (D) CFTR immunoblots and quantification in WT or F508del corrected at 30°C or by siHSF1 before (T = 0) and after brefeldin A treatment (BFA; 5 µg/ml) for the indicated time (h). The data is presented as a fraction of maximal band-C set to 1 at 1 h post-BFA treatment and represents the mean ± SD, <i>n</i>≥4. (E) Representative immunoblots from three experimental replicates of CFTR—WT, F508del at 37°C, F508del at 30°C, or F508del siHSF1—before and after proteolysis digestion with increasing concentration of trypsin (mg/ml). Upper blots show digestion pattern of CFTR NBD1 domain (18D1 antibody), and lower blots show digestion pattern of CFTR NBD2 domain (M3A7 antibody) (* indicates stable core fragment previously described) <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001998#pbio.1001998-Hoelen1" target="_blank">[75]</a>. (F) Quantification of FLuc activity in WT- and F508del-CFTR expressing cells treated with the indicated siRNA. The data represents specific FLuc activity (luminescence/relative FLuc expression) for each condition. Results are shown as a mean ± SEM, <i>n</i>≥3; * and # indicate <i>p</i><0.05 relative to WT- and F508del-CFTR, respectively. The underlying data used to make (A), (C), (D) and (F) in this figure can be found in the supplementary file <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001998#pbio.1001998.s008" target="_blank">Data S1</a>.</p