Exploring YAP1-centered networks linking dysfunctional CFTR to epithelial–mesenchymal transition

Cell lines

CFBE cells stably overexpressing wt- and F508del-CFTR (78) or overexpressing an inducible mCherry-Flag-CFTR construct (wt- and F508del-CFTR variants) (20), as well as Human Bronchial Epithelial Cell Lines (16HBE and 16HBEge) were used throughout this work.

CFBE wt-, F508del-CFTR 16HBE wt-, F508del-, and N1303K-CFTR cells were grown in Minimum Essential Medium Eagle (MEM) with Earl salts and L-glutamine (10-010-CVR; Corning) supplemented with 10% (vol/vol) FBS (10270; Gibco), 1% Pen/Strep (15070; Gibco), and 2.5 μg/ml puromycin (P8833; Sigma-Aldrich). 16HBE cells were seeded in 24-well plates at the density of 1.2 × 10⁵ cells/well. On the following day, the medium was changed from 10% to 2% (vol/vol) FBS, and 24 h later, protein was collected for subsequent WB experiment. To achieve polarization, CFBE cells were seeded on collagen IV pre-coated Transwell permeable supports at a density of 1.25 or 2.5 × 10⁵ cells, depending on the diameter of the filter (6.5 mm or 12 mm insert, Corning 3470 and 3460, respectively). On the following day, the medium was changed from 10% to 2% (vol/vol) FBS to promote differentiation/polarization. The transepithelial electrical resistance (TEER) was measured regularly using a volt-ohmmeter (Millicell-ERS, MER5000001; Millipore).

CFBE mCherry-Flag-CFTR cells were grown in DMEM with 4.5 g/l glucose and L-glutamine (BE12-604F; Lonza) supplemented with 10% (vol/vol) FBS, 10 μg/ml blasticidin (ant-bl-1; InvivoGen), and 2 μg/ml puromycin. 16HBE and 16HBEge cells were provided by CFF.

CFTR modulator treatments

3 μM VX-445 (S8851; SelleckChem), 5 μM VX-661 (S7059; SelleckChem), or 3 μM VX-809 (S1565; SelleckChem) were added to CFBE cells 48 h after transfection with NCtrl or siYAP1 (1) and left for 24 h. Compounds were dissolved in DMSO which was used as a negative control in the experiment.

Forward siRNA transfection

CFBE wt- and F508del-CFTR cells were grown to confluence and split (50%). After 24 h, the cells were seeded at an approximate density of 3.2 × 10⁴ cells.cm⁻². Transfection was performed 24 h later by mixing an siRNA solution with Lipofectamine 2000 (11668019; Invitrogen) at a 1:2 ratio in OptiMEM (31985088; Gibco), according to the manufacturer’s instructions (i.e., 1 μl of 30 μM siRNA—final concentration of 37.5 nM—with 2 μg of Lipofectamine 2000). Cells were grown in FBS-free media for 24 h before changing the media to MEM supplemented with 10% (vol/vol) FBS. At 72 h post-transfection, protein or RNA were collected.

16HBE-F508del-CFTR cells were seeded at a density of 1.4 × 10⁵ cells.cm⁻² on 24-well plates. Transfection was performed in suspension by mixing a siYAP1 or NCtrl solution with Lipofectamine 3000 (L3000015; Invitrogen) at a 1:0.75 ratio in OptiMEM, according to the manufacturer’s instructions (i.e., 1 μl of 30 μM siRNA—final concentration of 37.5 nM—with 0.75 μl of Lipofectamine 3000). 24 h post-transfection, the cells were treated with 3 μM VX-445 (S8851; SelleckChem) and 5 μM VX-661 (S7059; SelleckChem) for 24 h before protein was collected.

CFTR traffic screen

A high-throughput fluorescence microscopy pipeline for the assessment of the effects of particular genes on CFTR traffic has been previously developed (20). This assay relies on the CFBE mCherry-Flag-CFTR cells, which feature a mCherry tag at the N terminus of CFTR and a Flag tag in its fourth extracellular loop (Fig S6A), facilitating quantification of the total amount of CFTR and amount of CFTR at the PM, respectively. The pipeline allows the quantification (as a modified Z-score) of the impact of an individual gene on CFTR traffic by using siRNAs to KD that specific gene. Treatments are defined as “positive hits” or CFTR traffic enhancers if the siRNA increases PM-CFTR localization by Z > 1 and “negative hits” or CFTR traffic inhibitors if the siRNA decreases PM-CFTR localization by Z < −1. In the present work, a hypothesis-driven siRNA screen was performed with a library containing siRNAs targeting 30 genes known to be associated with EMT and (de)differentiation. The library contained 62 siRNAs: 59 targeted EMT and (de)differentiation genes (2 for most genes) and three were assay controls: a transfection control (siCFTR), a negative control (NCtrl, Negative Control #1, 4390843, Silencer Select; Ambion), and a positive control (siCOPZ1, targeting a subunit protein from COPI vesicles). Importantly, the positive control siCOPZ1 illustrates an F508del-CFTR traffic enhancer (Z = 1.62, Fig S6B and C), whereas treatment with siCFTR greatly decreased the fluorescence signal in almost all cells (Fig S6C), confirming a high-transfection efficiency. The library comprised a total of 83 different treatments, 62 with individual siRNAs, and 21 with two siRNA combinations. The combinations were performed to KD genes which have been reported to act together, for example, YAP1 and TEAD4, which are co-transcriptional activators. The full contents of the library are disclosed in Supplemental Data 1.

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Figure S6. CFTR traffic screen controls.

(A) The cystic fibrosis bronchial epithelial mCherry-Flag-wt- and F508del-CFTR cells used in the screen have a mCherry tag at the N terminus of CFTR and a Flag tag in its fourth extracellular loop. The mCherry fluorescence represents the total amount of CFTR and the Flag fluorescence represents the amount of CFTR at the plasma membrane (PM). (B) As controls of the screen, cystic fibrosis bronchial epithelial mCherry-Flag-CFTR cells were treated with siCOPZ1 and siCFTR, as well as with a non-targeting siRNA (NCtrl) and then stained and imaged. Immunofluorescence images were quantified to determine the CFTR PM levels in response to each siRNA treatment, which were then converted into a Z-Score. Data are presented as median ± SEM (n = 6). (C) Representative fluorescence microscopy images from the traffic screen controls. Traffic of mCherry-Flag-F508del-CFTR was enhanced by siCOPZ1 as represented by an increase in Flag fluorescence. Treatment with siCFTR greatly decreases CFTR expression, as seen by the decrease in mCherry (total CFTR) fluorescence, as well as Flag (PM CFTR) fluorescence (in wt-CFTR cells). In merged images, nuclei are depicted in blue, Flag (PM CFTR) in green, and mCherry (total CFTR) in red. Scale bar represents 50 μm.

Preparation of siRNA-coated 96-well plates for reverse transfection

96-well plates (655090; Greiner Bio-One) were coated with a custom siRNA library (Silencer Select, Ambion) for solid-phase reverse transfection in a protocol adapted from references 20 and 79. Initially, a transfection mixture of 1,200 μl was prepared by mixing 323 μl of Lipofectamine 2000, 554 μl of a 0.4 M sucrose in OptiMEM solution, and 323 μl of doubly distilled water. Separately, 5 μl of each siRNA at 3 μM added to each well of a conical bottom 96-well plate. Then, 7 μl of the transfection mix were added per well to the siRNA plate. After 20 min incubation at room temperature, 7 μl of 0.2% (wt/vol) gelatin solution were added to the siRNA plate. The total of 19 μl of the final mix (siRNA plus transfection mix plus gelatin) were diluted 1:50 in a 96-well deep well plate using double distilled water. Finally, 50 μl from the diluted mix were applied to each well of the 96-well imaging plates, which were lyophilized and stored in an anhydrous atmosphere. Careful mixing and centrifugation below 30g were performed between steps. This siRNA plate coating was performed with a manual 96-channel pipette (Liquidator96, 17010335; Mettler Toledo) and a VIAFLO electronic 96-channel pipette (6031; Integra).

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Figure S7. YAP1 immunofluorescence and respective negative controls.

Representative image of the negative controls from Fig 4F (i.e., secondary antibody staining without primary antibody). Images are shown as average intensity projections. In merged pictures nuclei are depicted in blue, the YAP1 channel in green and the ZO1 channel in red. Scale bar represents 10 μm (n = 3).

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Figure S8. Graphs with data points from Figs 2F, 3C, and 4E.

This representation was not used because most of the data are normalized to the negative controls, thus not adding information to the existing one. The only exception is Fig 4G where there is a greater distribution of the data points and thus these are shown on the graph.

CFTR traffic assay

CFBE mCherry-Flag-CFTR cells were grown to confluence and split (50%). After 24 h they were seeded in siRNA-coated 96-well plates (100 μl/well, 5 × 10³ cells) using a Multidrop Combi peristaltic dispenser (5840300; Thermo Fisher Scientific) and antibiotic-free medium. Reverse transfection occurred in the next 72 h. F508del-CFTR expression was induced in the last 48 h and wt-CFTR in the last 24 h of the transfection window, using 1 μg/ml doxycycline (D9891; Sigma-Aldrich).

Immunostaining

Cells were washed once with cold PBS++ (1× PBS supplemented with 1.1 mM MgCl₂ and 0.7 mM CaCl₂, pH 7.4) after which extracellular Flag-tags were immunodetected (see Table S1) without cell permeabilization for 1 h at 4°C. Cells were then incubated for 20 min at 4°C with 3% (vol/vol) PFA (104003; Merck Millipore), after which they were transferred to room temperature and incubated for 1 h with anti-mouse Cy5-conjugated secondary antibody (2 mg/ml, A10524; Invitrogen). Finally, the cells were incubated with a Hoechst 33342 solution (1 h, 200 ng/ml, B2261; Sigma-Aldrich) after which they were immersed in PBS++ and incubated overnight before imaging. Between incubations, cells were washed three times with PBS++ using the Tecan Hydrospeed plate washer (INSTHS-02; Tecan). PBS++ was used to prepare all solutions, with antibody solutions additionally containing 1% (wt/vol) BSA (A9647; Sigma-Aldrich). All liquid handling was performed with a Liquidator96 pipette.

Image acquisition and analysis

Cell imaging was performed at room temperature with an automated widefield Leica DMI 6000B system. A 10x HC PL APO objective (Leica) with a numerical aperture of 0.4 was used. Exposure times at maximum light brightness for Hoechst, mCherry, and Cy5 were 200, 1,300, and 8,000 ms, respectively. The Hoechst channel was used for contrast-based autofocus.

Automated image analysis was performed with open-source software tools (CellProfiler, R) and previously established pipelines for file management (80) and image quantification (20). At least five image fields in triplicate were analyzed for all treatments. Initially, images were background corrected with the dark frame/flat field procedure. Then, quality control (QC) procedures excluded cells which did not significantly express CFTR, had abnormal morphology (e.g., apoptotic cells), or contained a significant number of saturated pixels. Cell-wise fluorescence integration allowed quantifying the CFTR amount in the entire cell (mCherry) or PM (Cy5). Then, an additional QC step excluded plates in which CFTR expression was not decreased by at least 30% in the presence of siCFTR comparing with the NCtrl (i.e., transfection efficiency smaller than 30%). In each well, the median Cy5 fluorescence (PM-CFTR) was calculated and converted into a modified Z-score:Modified Z−score (Z)=x−MedianNCtrl,plateisdNCtrl,platei,where x is the median of the Cy5 integrated fluorescence of all cells at a given well, and MedianNCtrl,platei and sdNCtrl,platei are the median and SD fluorescence of wells containing the negative control siRNA. The modified Z-score is then averaged across experimental replicates and statistical significance versus the negative control is assessed with a t test. By definition, “positive hits” are the siRNAs increasing PM-CFTR localization by Z > 1 and “negative hits” are the siRNAs decreasing PM-CFTR localization by Z < −1.

WB

CFBE or 16HBE cells were washed twice with cold PBS and lysed with sample buffer containing 31.25 mM Tris HCl (30721; Sigma-Aldrich), pH 6.8; 1.5% (vol/vol) sodium dodecyl sulphate (SDS) (15553; Gibco); 10% (vol/vol) glycerol (92025; Sigma-Aldrich); 50 mM dithiothreitol (DTT) (D0632; Sigma-Aldrich), and protease inhibitor cocktail (11697498001; Roche). Benzonase (E1014; Sigma-Aldrich) 25 U/ml was also added to shear the DNA. 25–30 μg of protein were loaded onto polyacrylamide gels (4% for stacking and 7/10% for resolving gels) to perform SDS/PAGE. Transfer onto polyvinylidene difluoride (PVDF) membranes (IPVH00010; Merck Millipore) was performed using a wet-transfer system. The membranes were blocked for 1 h with 5% (wt/vol) non-fat milk (NFM) in PBS supplemented with Tween 20 (BP337-100; Fisher BioReagents). This was followed by incubation with the primary antibody overnight at 4°C, with gentle shaking. HRP-conjugated secondary antibodies were applied for 1 h at RT. All the antibodies were diluted in the blocking solution. Membrane luminescence was detected on a Chemidoc XRS+ system (170-8265; Bio-Rad). Quantification of band intensity was performed using the Image Lab software (170-9690; Bio-Rad), which integrates peak area. All measurements were normalized against loading controls (calnexin or GAPDH). A list of primary antibodies can be found in Table S1.

Semi-quantitative PCR (analysis of siRNA KD efficiency)

After siRNA transfection, RNA was extracted using the NZY Total RNA Isolation kit (MB13402; Nzytech) according to manufacturer’s instructions. cDNA was generated with NZY M-MuLV Reverse Transcriptase (MB08301; Nzytech). PCR reactions were then performed to evaluate the expression of the respective genes and of housekeeping control GAPDH. Each PCR reaction contained sense and antisense primers (0.2 μM), 20 ng cDNA and NZYTaq II DNA polymerase (MB354; Nzytech). The PCR reactions were carried out according to manufacturer’s instructions. cDNA was amplified during 35 cycles for 30 s at 94°C, 30 s at 54–58°C (depending on the primers) and 30 s at 72°C. PCR products were visualized by loading on RedSafe (21141; iNtROM) containing agarose gels. Membrane luminescence was detected on a Chemidoc XRS+ system. Quantification of band intensity was performed using the Image Lab software. All gene expression measurements were normalized against the respective GAPDH expression. Sequences for all primers are available in Table S2.

qRT-PCR

After CFBE cell polarization, RNA was extracted and cDNA was generated as described above. A mix containing forward and reverse primers, cDNA (5 ng) and 1x Evagreen SsoFast PCR reagent (172-5204; Bio-Rad) was used along with a Bio-Rad CFX96 system. Bio-Rad CFX Maestro 1.0 software was used for analysis. Mean relative transcript levels were calculated by normalizing the gene of interest against the control endogenous gene (GAPDH) and applying the ΔΔCT method where Fold Change (FC) = 2^−ΔΔCT. A standard cycle protocol was used for PCR amplification (1 min at 95°C followed by 40 cycles of 10 s at 95°C and 30 s at 60°C). Sequences for the primers were found at Harvard PrimerBank (Table S2). Melt curves confirmed amplification of unique specific products.

Patch-clamp

Patch-clamp experiments were performed to assess F508del-CFTR function after transfection with siYAP1 (1). CFBE F508del-CFTR cells grown on glass-coated cover slips were transfected with siYAP1 (1) and NCtrl for 72 h. The cover slips were then mounted on the stage of an inverted Zeiss microscope and kept at 37°C. Patch pipettes were filled with a cytosolic-like solution containing: KCl 30 mM, K-gluconate 95 mM, NaH₂PO₄ 1.2 mM, Na₂HPO₄ 4.8 mM, EGTA 1 mM, Ca-gluconate 0.758 mM, MgCl₂ 1.03 mM, D-glucose 5 mM, and ATP 3 mM, pH 7.2. Patch-clamp experiments were performed in the fast whole-cell configuration.

The bath was perfused continuously with Ringer solution: NaCl 145 mM, KH₂PO₄ 0.4 mM, K₂HPO₄ 1.6 mM, D-glucose 5 mM, MgCl₂ 1 mM, and Ca-gluconate 1.3 mM, pH 7.4, containing 50 nM TRAM34 (ab141885; Abcam) at a rate of 8 ml/min. Patch pipettes had an input resistance of 2–4 MΩ and whole cell currents were corrected for serial resistance. Currents were recorded using a patch-clamp amplifier (EPC 7, List Medical Electronics), the LIH1600 interface and PULSE software (HEKA) as well as Chart software (AD Instruments). In regular intervals, membrane voltage (Vc) was clamped in steps of 20 mV from −100 to +100 mV from a holding voltage of −60 mV. Current density was calculated by dividing whole-cell currents by cell capacitance. CFTR was activated with a combination of forskolin (2 μM) and genistein (25 μM).

Immunofluorescence staining of polarized cells (IF)

Polarized CFBE cells were fixed with PFA 4% (vol/vol), permeabilized with Triton X-100 (17-1315-01; Amersham Biosciences) 0.5% (vol/vol), and blocked with BSA 1% (wt/vol) before being removed from their supports using a scalpel. Cells were then incubated overnight at 4°C with primary antibodies, after which a mix of the secondary antibodies and nuclear dye (4 μg/ml, Methyl Green, 67060; Sigma-Aldrich) was applied for 2 h at RT. Negative controls were performed for each experiment by adding BSA instead of primary antibodies (Fig S7). Filter sections were mounted in a mix of N-propyl gallate (P3130; Sigma-Aldrich) and Glycerol for microscopy (104095; Merck). A list of primary antibodies can be found in Table S1.

Imaging was performed with a Leica TCS SP8 confocal microscope, using HC Plan Apo 20x/0.75 and HC Plan Apo 63x/1.4 objectives. Software used for acquisition was Leica's LAS x, and image processing was performed on ImageJ FIJI (81). FIJI was used to generate average image projections.

YAP1 fluorescence quantification

Image stacks were exported as TIF for fluorescence quantification. Nuclei were segmented using the Methyl Green channel. A median filter (radius = 7 × 7 × 2 pixels in xyz, or 0.5 × 0.5 × 0.6 μm) was applied and a manual threshold was applied. Object segmentation was refined with the 3D morphological opening implemented in the MorphoLibJ plugin (https://imagej.net/plugins/morpholibj, radius = 10 × 10 × 0 pixels or 0.7 × 0.7 × 0 μm). Then, nuclei were declumped using the 3D Watershed Split algorithm from the 3D ImageJ suite (https://imagej.net/plugins/3d-imagej-suite). Seeds were computed by performing ultimate points and 3D dilation (30 × 30 × 0 pixels or 2.1 × 2.1 × 0 μm) in MorphoLibJ. Manual editing was required to split some objects. The remaining cell cytoplasm was segmented by performing the “Dilate Labels” operation in MorphoLibJ (distance = 250 × 250 × 250 pixels or 17.7 × 17.7 × 74.6 μm). Cells touching the image border or containing small (<147 μm³), fragmented, mitotic, or otherwise unrepresentative nuclei were discarded. Finally, YAP1 fluorescence was integrated using the 3D ROI manager and the fraction of fluorescence localizing to the nucleus of each cell was calculated. Each image was summarized by the average nuclear fraction value of all of its cells. Differences in the average nuclear fraction were tested using t test at a significance of 0.05.

In vitro antibody agarose beads crosslinking

Throughout this work, all immunoprecipitations were preceded by an in vitro antibody agarose bead crosslinking step, to prevent excessively abundant peptides from the antibodies which can generate unwanted interferences. Antibodies were incubated with Pierce Protein G Agarose beads (20398; Thermo Fisher Scientific) at a 1:20 ratio (5 μl of antibody for 100 μl of beads) for 1 h rocking. Antibody-bound beads (henceforth referred to simply as beads) were then washed with sodium tetraborate decahydrate (S9640; Sigma-Aldrich), 0.1 M, pH 9, and resuspended again in sodium tetraborate decahydrate with dimethyl pimelimidate (DMP) (21667; Thermo Fisher Scientific) at the final concentration of 20 mM. The DMP and beads were mixed for 30 min at RT, after which the reaction was terminated by washing the beads once with ethanolamine (E0135; Sigma-Aldrich) 0.2 M pH 8 and three times with PBS. Finally, beads were resuspended in PBS with 0.02% (wt/vol) sodium azide (S2002; Sigma-Aldrich) and stored at 4°C until use (no longer than 48 h).

YAP1, CFTR, and TWIST1 co-immunoprecipitation

CFBE wt- and F508del-CFTR cells were washed three times with cold PBS, incubated in Triton lysis buffer (TBS) (25 mM Tris–HCl pH 7.4, 150 mM NaCl₂, 1% [vol/vol] and Triton X-100, supplemented with protease inhibitors) for 30 min rocking at 4°C, scraped and pelleted. For the immunoprecipitation, the supernatants were collected and pre-cleared for 1 h at 4°C with Protein G agarose beads, followed by incubation with anti-YAP1, anti-CFTR, or anti-TWIST1 crosslinked beads overnight at 4°C. Finally, the beads were washed twice with wash buffer (Tris–HCl 100 mM; NaCl₂ 300 mM) supplemented with 1% (vol/vol) Triton X-100 and twice with wash buffer without Triton. The beads were then resuspended in sample buffer (see above), after which WB was performed as described. Because this was a qualitative experiment, no quantifications were performed. A list of primary antibodies can be found in Table S1.

YAP1 immunoprecipitation and sample preparation for MS

YAP1 immunoprecipitation was performed as described. Briefly, cells were washed, incubated in TBS, scrapped, and pelleted. Supernatants were then pre-cleared and incubated with anti-YAP1 crosslinked beads, which were washed and resuspended in sample buffer. Approximately 10 μg of protein were then loaded onto polyacrylamide gels (4% for stacking and 10% for resolving gels) to perform a short gel SDS/PAGE (proteins ran for about 1 cm in the 10% gels). Gels were then washed with ultrapure water to remove the SDS and incubated with PageBlue Protein Staining Solution (24620; Thermo Fisher Scientific) for 1 h with agitation. Gels were washed again with ultrapure water and each stained lane (corresponding to one immunoprecipitation sample) was excised and cut into ∼1 mm³ cubes, which were stored in Protein LoBind Tubes (0030108116; Eppendorf) in ultrapure water. Samples were kept at 4°C until nano-liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) analysis was performed. Gel bands were then excised, reduced in 10 mM DTT (Sigma-Aldrich) for 40 min at 56°C, and alkylated in 55 mM iodoacetamide (Sigma-Aldrich) for 30 min in the dark. The resulting sample was digested overnight with trypsin (Promega) at 37°C and cleaned up with a C18 column (OMIX C18; Agilent).

NanoLC–MS/MS analysis and protein identification

NanoLC–MS/MS analysis was performed on an ekspert NanoLC 425 cHiPLC system coupled with a TripleTOF 6600 with a NanoSpray III source (Sciex). Peptides were separated through reversed-phase chromatography (RP-LC) in a trap-and-elute mode. Trapping was performed at 2 μl/min on a Nano cHiPLC Trap column (Sciex 200 μm × 0.5 mm, ChromXP C18-CL, 3 μm, 120 Å) with 100% A for 10 min. The separation was performed at 300 nl/min, on a Nano cHiPLC column (Sciex 75 μm × 15 cm, ChromXP C18-CL, 3 μm, 120 Å). The gradient was as follows: 0–1 min, 5% B (0.1% formic acid in acetonitrile; Fisher Chemicals); 1–46 min, 5–35% B; 46–48 min, 35–80% B; 48–54 min, 80% B; 54–57 min, 80–5% B; 57–75 min, and 5% B.

Peptides were sprayed into the MS through an uncoated fused-silica PicoTip emitter (360 μm O.D., 20 μm I.D., 10 ± 1.0 μm tip I.D., New Objective). The source parameters were set as follows: 15 GS1, 0 GS2, 30 CUR, 2.5 keV ISVF, and 100°C IHT. An information dependent acquisition (IDA) method was set with a TOF-MS survey scan of 400–2,000 m/z. The 50 most intense precursors were selected for subsequent fragmentation and the MS/MS were acquired in high sensitivity mode for 40 msec. The obtained spectra were processed and analyzed using ProteinPilot software, with the Paragon search engine (version 5.0; Sciex). A UniProt reviewed database (20,394 entries, accessed on 05/01/2021) containing the sequences of the proteins from Human was used. The following search parameters were set: Iodoacetamide, as Cys alkylation; Trypsin, as digestion; TripleTOF 6600, as the Instrument; gel-based ID as Special factors; Biological modifications as ID focus; Search effort, as thorough; and an FDR analysis. Only the proteins with Unused Protein Score above 1.3 and 95% confidence were considered.

GO analysis and GSEA

Available bioinformatic tools were used to analyze the lists of YAP1 interactors found in the wt- and F508del-CFTR CFBE cells. Data were submitted to the PANTHER (Protein ANalysis THrough Evolutionary Relationships) Classification System (82) to generate a list of the enriched GO biological process (BP) terms (P < 0.05). These were then manually curated and grouped into 11 different categories that included most of the enriched BP terms: apoptosis, barrier function, cell cycle, cell junction, cytoskeleton, differentiation, ECM, immune system, inflammation, miRNA, and stress. Two figures were constructed, one representing all the enriched (fold enrichment > 15) BP terms found for the YAP1 interactors on both cell lines (Fig S4B), and another showing the enriched (fold enrichment > 8.5) differential BP terms found for each cell line (Fig 4B).

Data were also submitted to GSEA (83), particularly to the Molecular Signatures Database (MSigDB) to identify gene sets differentially expressed by the YAP1 interactors in the presence of wt- or F508del-CFTR (P < 0.05). Data were analyzed regarding hallmark gene sets (MSigDB gene sets that represent well-defined biological states or processes) as well as oncogenic signature gene sets (generated from microarray gene expression data from cancer gene perturbations).

PPI networks

To assemble a novel PPI network bridging (dysfunctional) CFTR and EMT through YAP1, a subset of genes/proteins of interest derived from the MS analyses, was established. This subset consisted of genes/proteins that met at least one of the following criteria, namely, if they were (ii) F508del-CFTR-specific YAP1 interactors defining unique GO BP terms related to CF and/or EMT, that is, from the manually curated biological categories ECM and inflammation (see Supplemental Data 3); (ii) F508del-CFTR–specific YAP1 interactors defining gene sets enriched in F508del-CFTR cells and related to CF and/or EMT, that is, MYC signaling, EMT, mitotic spindle (mitosis), hypoxia, and TGFβ signaling (see Supplemental Data 4); and (iii) F508del-CFTR-specific YAP1 interactors that were unique oncogenes or defined oncogenic signatures in F508del-CFTR cells (see Supplemental Data 5). This subset comprised 65 proteins (Fig 6A), excluding YAP1 and CFTR. To investigate which, out of these interactors, were the more relevant in the context of CF, they were filtered by one of two criteria: (i) proteins that appeared in more than one category (represented by “*“ in Fig 6A) and/or (ii) proteins that are part of the published CFTR interactome (27) (represented by the different colors in Fig 6A). This list of proteins of interest from the YAP1 interactome comprised 30 high confidence proteins (Fig 6A), for which predictions of PPI networks were generated using the STRING database (28) (medium level of confidence). STRING integrates all publicly available sources of PPI information and complements these with computational predictions.

Antibodies, primers, and siRNAs

Information regarding the siRNAs used in the present study are available in Supplemental Data 1. A list of primary antibodies used in both IF and WB can be found in Table S1. Sequences for the primers used are listed in Table S2.

Statistical analyses

Data are presented as mean ± SEM, mean ± SD, and median ± SEM, as indicated in the figure legends. Graphical representation in the form of data points was not pursued since most of the data was normalized to the control condition (Fig S8). Number of replicates is also present in all figure legends and indicates how many times the experiment was performed (i.e., n = 3 indicates experiments performed in triplicate). t test for unpaired samples was performed for statistical analyses. Either Prism 6 software (GraphPad, Inc.) or open-source software R were used for graph design and statistical analyses. R-based statistical analysis is described in the “Image acquisition and analysis” sections. R-based graph design was performed with the ggplot2 (84) and VennDiagram (85) packages. Significant differences were defined for P < 0.05.