c-Cbl, CHIP or Nedd4-2 depletion do not affect rΔF508 CFTR endocytosis.

<p>CFBE41o-ΔF cells were transfected with control or c-Cbl, CHIP, or Nedd4-2 siRNA oligonucleotides. At 24 h after transfection, cells were transferred to Transwell filters and cultured for an additional 4–5 days. The last 24 h, the cells were cultured at 27°C to promote ΔF508 CFTR delivery to the cell surface. <b>(A)</b> Cell surface expression of rΔF508 CFTR after siRNA transfection of control (C), c-Cbl, CHIP or Nedd4-2 as indicated. The knockdown efficiency of c-Cbl, CHIP, and Nedd4-2 was >95%. β-actin was blotted as a loading control. <b>(B)</b> Representative blots showing the remaining surface rΔF508 CFTR after 2.5 min internalization at 37°C. <b>(C)</b> Quantitative analysis of rΔF508 CFTR internalization after 2.5 min warm-up following c-Cbl, CHIP, or Nedd4-2 siRNA depletion and low-temperature rescue. The rate of rΔF508 CFTR internalization was measured after 2.5 min warm-up as described in the Material and Methods section. Depletion of c-Cbl, CHIP, or Nedd4-2 had no significant (N.S.) effect on rΔF508 CFTR internalization (n = 3).</p

Increased cell surface expression of rescued ΔF508 CFTR (rΔF508 CFTR) following μ2 and Dab2 depletion.

<p>The consequences of μ2 depletion and Dab2 depletion on total ΔF508 CFTR expression (<b>A</b> and <b>B</b>) and on surface rΔF508 CFTR expression (<b>C</b> and <b>D</b>). CFBE41o-ΔF cells were transfected with 20 nM siRNA duplexes targeted specifically to μ2 or Dab2 or control (C) siRNA. 48 h after transfection, the control and siRNA depleted cells were cultured for an additional 24 h at 27°C to promote ΔF508 CFTR delivery to the cell surface. 72 h after transfection, 25 μg of cell lysates were separated by SDS-PAGE and immunoblotted using anti-CFTR, anti-μ2 and anti-Dab2 antibodies. β-actin was blotted as loading control. The changes in μ2, Dab2 and rΔF508 CFTR levels following siRNA depletion are indicated below the blots. Cell surface expression of rΔF508 CFTR was monitored by biotinylation as described in the Experimental section. The molecular mass in kDa is indicated on the right-hand side of each panel.</p

Increased transepithelial chloride transport following Dab2 depletion in CFBE41o-ΔF monolayers.

<p>CFBE41o-ΔF cells were transfected with control or Dab2 siRNA oligonucleotides. At 24 h after transfection, cells were lifted, seeded on to Transwell filters and cultured for an additional 4–5 days. The last 24 h, the cells were cultured at 27°C to promote ΔF508 CFTR delivery to the cell surface. The <i>I</i>_<i>SC</i> across the monolayers was measured in Ussing chambers as described in the Experimental Section. <b>(A)</b> Representative tracings from control and Dab2-depleted monolayers. After a stable baseline was attained, 20 μM forskolin (F), 50 μM genistein (G) and 10 μM CFTR_inh-172 were added at the indicated arrows. <b>(B)</b> Forskolin and genistein activated <i>I</i>_<i>SC</i>. <i>ΔI</i>_<i>SC</i> was calculated as an increase in <i>I</i>_<i>SC</i> after forskolin and genistein addition over the base-line currents (n = 4). <b>C.</b> CFTR_inh-172 inhibited <i>I</i>_<i>SC</i>. <i>ΔI</i>_<i>SC</i> was calculated as a decrease in <i>I</i>_<i>SC</i> after CFTR_inh-172 addition.</p

CHIP depletion increases the surface half-life.

<p>CFBE41o-ΔF cells were transfected with control or c-Cbl <b>(A)</b>, CHIP <b>(B),</b> or Nedd4-2 <b>(C)</b> siRNA oligonucleotides and cultured for 48 h at 37°C. The cells were then transferred to 27°C and incubated for an additional 48 h. Protein synthesis was stopped by preincubation with cycloheximide during the last 2 h at 27°C. Fresh media containing cycloheximide (CHX) was added and the cells were incubated for 0 to 6 h at 37°C and the surface pool of rΔF508 CFTR was detected using biotinylation as described in the Material and Methods section. Quantitative analysis of the blots is shown at the bottom (n = 3). The CHIP depletion significantly increased the cell surface half-life of rΔF508 CFTR, whereas c-Cbl depletion had little and Nedd4-2 depletion had no effect. <b>(D)</b> Cell surface half-life of rΔF508 CFTR was analyzed after CHIP depletion in combination with c-Cbl and/or Nedd4-2 as indicated. Depletion of c-Cbl and Nedd4-2 in combination with CHIP depletion did not have additional effect on the surface half-life of rΔF508 CFTR.</p

CHIP Depletion increases the function of rΔF508 CFTR.

<p>CFBE41o-ΔF cells were transfected with control or CHIP siRNA oligonucleotides, seeded on to Transwell filters and the <i>I</i>_<i>SC</i> across the monolayers was measured in Ussing chambers as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123131#pone.0123131.g004" target="_blank">Fig 4</a> legend. <b>(A)</b> Representative tracings from control and CHIP-depleted monolayers. After a stable baseline was attained, 20 μM forskolin (F), 50 μM genistein (G) and 10 μM CFTR_inh-172 were added at the indicated arrows. <b>(B)</b> Forskolin and genistein activated <i>I</i>_<i>SC</i>. <i>ΔI</i>_<i>SC</i> was calculated as an increase in <i>I</i>_<i>SC</i> after forskolin and genistein addition over the base-line currents. <b>C.</b> CFTR_inh-172 inhibited <i>I</i>_<i>SC</i>. <i>ΔI</i>_<i>SC</i> was calculated as a decrease in <i>I</i>_<i>SC</i> after CFTR_inh-172 addition. (n = 6)</p

VX-809 stabilizes the cell-surface pool of rΔF508 CFTR.

<p><b>(A)</b> ΔF508 CFTR in CFBE41o-ΔF cells were rescued to cell surface by incubating at 27°C for 48 h followed by 3 h pretreatment with 3 μM VX-809. The cells were transferred to fresh medium containing cycloheximide (CHX) with or without VX-809 that is prewarmed to 37°C and incubated at 37°C for the time periods indicated. The rescued ΔF508 CFTR (rΔF508) remained on the cell surface was measured by biotinylation as described in the Methods section. (B) The ubiquitination of rΔF508 CFTR were determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123131#pone.0123131.g009" target="_blank">Fig 9</a> legend. Treatment with 3 μM VX-809 inhibited the ubiquitination of rΔF508 CFTR.</p

rΔF508 CFTR delivery to the late endosomes is inhibited in Dab2-depleted cells.

<p>CFBE41o-ΔF cells were treated with control <b>(A</b> and <b>B)</b> or Dab2-specific <b>(C)</b> siRNA oligonucleotides. At 24 h after transfection, the cells were cultured at 27°C for an additional 48 h to facilitate cell surface delivery of ΔF508 CFTR. After the low-temperature rescue, one set of the control cells was transferred to 37°C for 1 h (A), another set of the control (B) and the Dab2-depleted cells (C) were treated with 5 mM ammonium chloride for 1 h at 37°C followed by immunofluorescent staining of CFTR and M6PR. <b>(A) r</b>ΔF508 CFTR and mannose-6-phosphate receptor (M6PR) do not co-localize in control untreated cells. <b>(B)</b> Ammonium chloride treatment (inhibition of lysosomal degradation) resulted in rΔF508 CFTR and M6PR co-localization (right-hand panel, arrowheads)<b>. (C)</b> Dab2 depletion and ammonium chloride treatment together enhanced rΔF508 CFTR staining; however, no co-localization of rΔF508 CFTR and M6PR is apparent.</p

Reduced endocytosis rates of rΔF508 CFTR following μ2 or Dab2 depletion.

<p>CFBE41o-ΔF cells were transfected with control, μ2 or Dab2 siRNA oligonucleotides as indicated. At 24 h after transfection, the cells were transferred to Transwell filters and incubated for an additional 4–5 days under an air-liquid interface. During the last 24 h, the cells were incubated at 27°C to promote ΔF508 CFTR rescue. The efficiency of μ2 and Dab2 depletion was >90%. CFTR internalization assays were performed as described previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123131#pone.0123131.ref028" target="_blank">28</a>]. <b>(A)</b> Representative gels of CFTR internalization assays. The molecular mass in kDa is indicated on the right-hand side. <b>(B)</b> Quantitative analysis of rΔF508 CFTR internalization rates during a 7.5 min time period. The percentage of internalized CFTR was calculated from the loss of biotinylated CFTR during a 37°C incubation for time periods indicated after comparing to that at time 0 min under each condition (n = 3). <b>(C)</b> Quantitative analysis of CFTR internalization rates after 37°C warm-up for 2.5 min following μ2 or Dab2 depletion. Depletion of μ2 or Dab2 significantly reduced CFTR internalization rates in a 2.5 min time period (n = 3, *p<0.05).</p

Increased cell surface half-life of rΔF508 CFTR in μ2- and Dab2-depleted cells.

<p>CFBE41o-ΔF cells were transfected with control, μ2, or Dab2 siRNA oligonucleotides. 48 h after transfection, the cells were cultured for 24 h at 27°C to allow cell-surface expression of rΔF508 CFTR. Cell surface rΔF508 CFTR was then monitored by biotinylation as described in the Material and Methods section after incubating with cycloheximide (CHX)-containing medium at 37°C for time periods indicated. Representative gels are shown (<b>A</b>) and quantitative analysis of the half-lives of rΔF508 CFTR under each experimental condition is shown (<b>B</b>). Dab2 depletion resulted in a ~2 fold increase in the half-life of cell surface rΔF508 CFTR (n = 3).</p

Additional file 6: Table S3. of Codon bias and the folding dynamics of the cystic fibrosis transmembrane conductance regulator

CADD analysis of CFTR SNPs. SNPs that may affect splicing are marked bold. SNPs with C-score significantly higher than whole genome median are marked light grey, while SNPs with highest quartile C-scores are marked dark grey. (XLSX 28 kb