ML141 selectively reduces the GTP-bound form of Cdc42.

<p>Before lysis, cells were submitted to 10 μM ML141 for 30 min and 1% DMSO (v/v) was used as the control condition. After enrichment of the activated forms of GTPases in the clarified lysates, GST-pull-down was performed. Cdc42, Rac1 or RhoA protein amounts were then assessed in the resulting samples. (A) Representative Western blot images are shown. Densitometric quantification of bands was normalized to DMSO condition. (B) Histogram displays relative activated GTPase amounts, expressed as the percentage of control. Data represent means ± SEM of 3 independent experiments each performed in duplicate. *: p<0.05, ns: non significant.</p

Cdc42 depletion does not alter fibrillar actin content.

<p>RNAi-mediated depletions of Cdc42 or N-WASP were performed for 48 h and F-actin content was quantified as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118943#pone.0118943.g005" target="_blank">Fig. 5</a> legend. As displayed in histograms, N-WASP depletion alone elicited a decrease of polymerized actin content, compared with negative control RNAi condition. Data represent means ± SEM of 3–8 independent experiments, each performed in triplicate. **: p<0.01, ns: non-significant.</p

Cdc42 depletion increases CFTR stability at plasma membrane.

<p>Two approaches were used to assess CFTR turnover downstream of its targeting to plasma membrane. (A) Cdc42 depletion increases stability of the PM-targeted CFTR. The upper diagrams summarize the procedures followed. 48 h after cell transfection by the mentioned siRNA, surface proteins were labelled. Streptavidin capture occurred either subsequently (0), or after a 24 h incubation of cell cultures at 37°C (24). Labelled CFTR protein amounts were assessed in the resulting samples. After densitometric quantification of Western blot images (representative examples in the bottom left panel), (24) to (0) ratios were calculated. The Ctrl RNAi value was used to define 100% of labelled CFTR stability. In the bottom right panel, the histogram shows that the relative PM-CFTR stabilities increase when Cdc42 is depleted. (B) Cdc42 depletion increases remaining PM-CFTR after cycloheximide chase. The upper diagrams summarize the procedures followed. 48 h after cell transfection by siRNA, the PM proteins were labelled and purified (-). Alternatively, the cell cultures were submitted to additional 24 h incubation with 100 μg/mL cycloheximide (+), before biotinylation and capture were performed. The labelled CFTR protein amounts were assessed by densitometric quantification of Western blot bands (representative images in the bottom left panel). In both RNAi conditions, labelled CFTR amounts extracted from the same amount of whole cell lysates appeared higher after 24h CHX chase: this could be explained by stability differences between the various cellular proteins. To overcome this bias, we compared (+) to (-) ratios and the Ctrl RNAi value was used to define 100% of CFTR apparent PM stability. In the bottom right panel, histogram shows that CFTR stability at PM appears higher when Cdc42 is depleted. Data represent means ± SEM of 3 independent experiments, each performed in duplicate. ***: p<0.001.</p

Impact of Cdc42 or N-WASP depletions upon CFTR amount and internalization.

<p>RNAi-mediated depletions of Cdc42 or N-WASP were performed for 48 h. We then used biotinylation experiments to evaluate CFTR amount at the cell surface and CFTR internalization, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118943#pone.0118943.g002" target="_blank">Fig. 2</a> legend. (A) Representative Western blots and (B-D) histograms summarizing the data are presented. Following Cdc42 depletion, (B) total CFTR amount in whole cell lysate, (C) as well as CFTR amount at the plasma membrane, had increased compared to negative control RNAi conditions. (D) Internalized CFTR amount had decreased following Cdc42 depletion. Data represent means ± SEM of 3 independent experiments, each performed in duplicate. ***: p<0.001, **: p<0.01.</p

ML141 or wiskostatin treatments stimulate CFTR endocytosis.

<p>(A) Representative Western blots and (B and C) histograms summarizing the data are presented. First, cells were submitted to 10 μM ML141 for 30 min or 10 μM wiskostatin for 120 min. Treatment with 1% DMSO (v/v) was used as a negative control. Surface proteins were then biotinylated. Total CFTR protein amount was assessed by immunoblot in denatured samples obtained from 20 μg of clarified lysates (α1 Na⁺/K⁺ ATPase was used as a normalization control). Biotinylated proteins were purified from 100 μg of clarified lysates and the amount of labelled CFTR was assessed in the resulting samples. (B) The amount of plasma membrane CFTR, expressed as the percentage of DMSO treatment condition, decreased following both pharmacological treatments. Alternatively, the biotinylated PM proteins were allowed to enter the inner cell compartment through 5 min incubation of cell cultures at 37°C. Surface-exposed biotin moieties were then stripped by MESNA reduction. Biotinylated (internalized) proteins were purified from 600 μg clarified lysates and analyzed by Western blot. The ratio of the densitometric quantification of bands to the relative initial PM-CFTR amounts was then calculated. (C) Relative CFTR internalization, expressed as the percentage of DMSO control condition, appeared to have increased following ML141 or wiskostatin treatments. Data represent means ± SEM of 3 independent experiments, each performed in duplicate. ***: p<0.001, **: p<0.01, *: p<0.05.</p

Alteration of dynamin 2-dependent mechanisms impairs the stimulation of CFTR endocytosis by ML141 or wiskostatin treatments.

<p>CFBE-wtCFTR cells were transfected with the corresponding siRNA to deplete dynamin 2 or caveolin 1 proteins 48 h before performing quantification of PM-CFTR or of internalized CFTR, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118943#pone.0118943.g002" target="_blank">Fig. 2</a> legend. Histograms summarizing analyzed data are displayed. Results were compared with negative control siRNA transfection conditions. (A) Dynamin 2 depletion elicited an increase of PM-CFTR amount, (B) correlated to lower CFTR internalization, (A and B) whereas caveolin 1 depletion had no impact upon PM- and internalized CFTR amounts. Then, 48 h after transfection with indicated siRNA, cells were treated with 10 μM ML141 for 30 min or 10 μM wiskostatin for 120 min, before labelling and during the 5 min internalization period. Results were compared to vehicle treatment conditions. (C) When dynamin 2 is depleted, CFTR internalization increase upon pharmacological treatments is abolished. Data represent means ± SEM of 3 independent experiments, each performed in duplicate. ***: p<0.001, **: p<0.01, *: p<0.05, ns: non significant.</p

Pharmacological inhibitors of Cdc42 pathway impair CFTR channel activation.

<p>(A) Iodide efflux curves obtained in CFBE-wtCFTR cells treated with 10 μM wiskostatin for 120 min, 10 μM ML141 for 30 min or corresponding vehicle, prior to stimulation of CFTR activity by forskolin (Fsk, 10 μM) + genistein (Gst, 30 μM), n = 4 in each condition. (B) Histograms show the mean relative rates of CFTR activity. The result obtained with wiskostatin (resp. ML141) was compared with DMSO 1/1000 (resp. DMSO 1/100) (v/v) treatment. Means ± SEM are indicated. ***: p<0.001, *: p<0.05, ns: non significant.</p

Analysis of CFTR maturation and turnover by metabolic labelling.

<p>CFBE-wtCFTR cells were transfected with negative control or Cdc42 siRNA and cultured 48 h prior to pulse-chase experiments. Cells were pulse-labelled for 15 min with 100 μCi/mL of [³⁵S]methionine and [³⁵S]cysteine mix and then chased for 0, 0.5, 1, 2, and 4 h. CFTR was then immunoprecipited and subjected to SDS-PAGE. Bands corresponding to core-glycosylated (band B) and fully-glycosylated (band C) CFTR were quantified by densitometry for analysis. (A) Representative gels are shown. (B) Maturation of CFTR is evaluated as the ratio of band C detected at a given time relative to total CFTR (bands B+C). (C) CFTR turnover is displayed as the relative total CFTR amount (bands B+C) along the chase. Total CFTR amount is assigned a value of 100 in arbitrary units at the beginning of the chase (0 h), when the cells are transfected with negative control siRNA. (D) The rate of CFTR disappearance is estimated as the natural logarithm of the amount of CFTR (bands B+C) at a given time of chase relative to its amount at the beginning of the experiment (B₀+C₀). Displayed lines are the linear regressions to the data. Symbol and error bars are means ± SEM of the values at each point. The numbers of independent experiments used to build the graphs are indicated on the figures.</p

Cdc42 depletion increases CFTR channel activation.

<p>(A) In non-stimulated CFBE-wtCFTR cells (basal) or upon stimulation of CFTR activity by forskolin (Fsk, 10 μM) + genistein (Gst, 30 μM), iodide efflux curves were obtained after 48 h of siRNA-mediated depletions (negative control, Cdc42 or N-WASP), n = 4 in each condition. (B) Histograms show the mean relative rates of CFTR activity. Results obtained after Cdc42 and N-WASP depletions were compared to control RNAi condition. Means ± SEM are indicated. **: p<0.01, *: p<0.05.</p

Pharmacological treatments decrease actin polymerization.

<p>(A) Cell shape, polarity and peripheral actin cytoskeleton pattern appear preserved upon 10 μM wiskostatin or 10 μM L141 treatments compared to vehicle treatment, whereas actin is scattered throughout the cytoplasm with 100 μM wiskostatin. Cells were grown on Transwell permeable support until a monolayer was established. After ML141 or wiskostatin treatments at the indicated concentrations, ZO-1 proteins were immunostained (green) and actin was tracked using phalloidin-TRITC (red). TO-PRO-3 was used as a cell nucleus marker (blue). Transversal section images were acquired with a confocal microscope. Scale bars represent 20 μm. (B) Pharmacological inhibitions of Cdc42 pathway reduce fibrillar (F-) actin content. Cells were incubated with 10 μM ML141 or 10 μM wiskostatin, and 10 μM cytochalasin B was used as an F-actin polymerization inhibition positive control treatment, whereas 1% DMSO (v/v) was used as negative control treatment. Actin-bound phalloidin-FITC was methanol-extracted and fluorescence measurements were performed. Results were normalized to protein amount and the relative F-actin contents are expressed as the percentage of DMSO control condition in histograms. Data represent means ± SEM of 3 independent experiments, each performed in triplicate. **: p<0.01, *: p<0.05.</p