Hvin 1

Cells were cultured on glass coverslips, washed with PBS and fixed with 4% PFA. Fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5′ and moved to 0.2% Dulbecco/BSA. Immunofluorescence stainings were performed as described previously (26 (link)) and were repeated at least three times. The following primary antibodies were used in stainings: anti-E-cadherin (#14472, Cell Signaling Technology, Inc., Danvers, MA, USA), anti-vimentin (#5741, Cell Signaling Technology, Inc., Danvers, MA, USA), and anti-vinculin antibody (1:50) (hVin-1, Sigma, Saint Louis, MO, USA). The following secondary antibodies were used to detect the primary antibodies: Alexa Fluor α-rabbit 488 and α-mouse 568 (Life Technologies™, Carlsbad, CA, USA). Other reagents: Alexa-488- and -647-Phalloidins were used to visualize actin cytoskeleton in 1:200 dilution (Life Technologies™, Carlsbad, CA, USA), DAPI for DNA (Life Technologies™, Carlsbad, CA, USA), and DABCO/Mowiol was used in mounting. The images were acquired with Leica DM6000 upright fluorescence wide field microscope equipped with Hamamatsu Orca-Flash4.0 V2 sCMOS camera.

MDCK cell islands at 3, 6, 9 and 12 hours after releasing were pre-treated with 0.4% Triton X-100/0.4% paraformaldehyde in PBS for 3 min and fixed with 4% paraformaldehyde in PBS for 20 min. Cells were permeabilized with 0.4% Triton X-100 and blocked with 5% bovine serum albumin (BSA). The antibody for E-cadherin (24E10; 1:50; Cell Signaling) or Vinculin (hVIN-1; 1:400; Sigma-Aldrich) was treated to the cell islands. After washing the cells with PBST, Texas Red-conjugated secondary antibody, Alexa Fluor 488 phalloidin (1:200; Life Technologies) and Hoechst (33342; 1:3000; Life Technologies) were added for staining the cell islands. Cells were mounted in Elvanol reagent and covered with a cover glass (diameter = 12 mm; Marienfeld). Fluorescent images were taken with using a fluorescence microscope (Axioscope; Carl Zeiss) and a confocal microscope (LSM 510, Carl Zeiss). The confocal images were captured ~15 images at 100 nm interval per a position. In order to observe representative regions for focal adhesion and adherens junction, 4 images at the basal sections (0.1~0.4 μm) and in the mid-apical sections (0.9~1.2 μm) were respectively merged by projections of maximal intensity using ImageJ.

Cells were fixed in 4% PFA for 10 minutes and permeabilized with 0.2% Triton-X100. Cells were blocked for 45 min in 2% bovine serum albumin and incubated with primary antibody overnight at 4 °C. Primary antibodies used were mouse anti-vinculin (1:200, hVIN-1, Sigma Aldrich), mouse anti-β1 integrin (1:200, P5D2, Cancer Research UK, London, UK), rabbit anti-phospho-myosin light chain (1:100, Cell Signaling, Danvers, MA), and rabbit anti-Importin 9 (1:100, Abnova, Taipei, Taiwan). Coverslips were washed and incubated for 60 minutes with goat anti-mouse or goat anti-rabbit secondary antibody Alexa Fluor^® 568 (1:1000, Thermo Fisher Scientific) and DAPI. Alexa Fluor 568- or 633-phalloidin (Thermo Fisher Scientific) staining was used to visualize F-actin. Imaging was performed with a Zeiss 710 confocal microscope.

Cells were fixed with 4% PFA, washed 3 x with 0.2% Dulbecco/BSA and permeabilized with 0.1% Triton X-100 in TBS. Immunofluorescence stainings were performed as in (Tojkander et al., 2011 (link)). Images were acquired with a charge-coupled device camera (AxioCam HRm; Zeiss) on a microscope (Axio Imager.M2; Zeiss). AxioVision Rel. 4.8 (Zeiss) and PlanApo 63x/1.40 (oil) objective (Zeiss) was used for the image acquirement. The following reagents and antibodies were used for the stainings: Alexa phalloidin 488, 568, 594 and 647 (1:200–400 dilutions) (Life Technologies^TM), anti-cofilin-1 antibody (Abcam, ab11062), anti-VASP antibodies (1:50–100) (Sigma, HPA005724 and Enzo, IE273), VASP-phospho-T278 antibody (1:50) (ImmunoWay), VASP-phospho-S239 antibody (1:50) (Millipore, 16C2), anti-vinculin antibody (1:50) (Sigma, hVin-1). DAPI and secondary antibodies, which were conjugated to Alexa Fluor 488, Alexa Fluor 568/594, or Cy5 were from Life Technologies.

COS7 cells seeded at 30% confluency were transfected with the following siRNA: Control siRNA (mismatched control duplexes, Ambion); Arp2/3 siRNA1 (5′-AAAUCCUAAUGGAGACAAA-3′, Ambion), Arp2/3 siRNA2 (5′-CAUCACGGUUGGAACGAGAACUUAA-3′, Ambion); Vinculin siRNA1 (5′-GCUUCAAUCAAAAUUCGAA-3′, Ambion), Vinculin siRNA2 (5′-GGCUGCGGUUGGUACUGCUAAUAAA-3′, Ambion); integrin-β1 siRNA1 (5′-CCGUAGCAAAGGAACAGCA-3′, Ambion), integrin-β1 siRNA2 (5′-CCUAAGUCAGCAGUAGGAACAUUAU-3′, Ambion). siRNA transfection was performed at a final concentration of 75 nM using siPORT Amine transfection agent (Ambion) according to the manufacturer’s instructions. The efficiency of silencing was assessed at 3 days after transfection by western blot using antibodies against Arp2 (N1C3, 1:2000 dilution of 1 mg/ml stock; Genetex), integrin-β1 (12G10, 1:1000 dilution of 1 mg/ml stock; abcam), vinculin (hVIN-1, 1:2000 dilution of 1 mg/ml stock; Sigma) or β-tubulin (T4026, 1:5000 dilution of 2.6 mg/ml stock; Sigma).

Protein extracts were obtained from control lungs, ADC and PSC tumors. Total protein extracts (50 µg) from each sample were subjected to SDS-PAGE and transferred to nitrocellulose membranes (Amersham Biosciences, Arlington Heights, IL, USA). Membranes were blocked in PBS (Phosphate-buffered saline) containing 5% BSA (bovine serum albumin) and immunodetection was perform using antibodies against phospho-Akt (Ser473) (D9E) (Cell Signalling Technology, Danvers, MA, USA), Akt (pan) (C67E7) (Cell Signalling Technology, Danvers, MA, USA) and vinculin (hVIN-1) (Sigma-Aldrich, San Luis, MO, USA). In all cases, membranes were incubated with a horseradish peroxidase (HRP)-labeled secondary antibody and detected by luminography using Immobilin Western Chemiluminescent HRP Substrate (Millipore, Burlington, MA, USA).

A375‐Cas9, IGR1‐Cas9, and WM983B‐Cas9 cells were transduced with lentiviral sgRNA expression vectors and cell lysates were prepared for immunoblot analysis as described previously (Christodoulou et al., 2019). Antibodies used for detection included anti‐DUSP4 (1:1,000), anti‐PPP2R2A (1:1,000), anti‐BRAF (1:1,000) (Cell Signaling Technology), and anti‐Vinculin (1:1,000, clone hVIN‐1, Sigma‐Aldrich) as a loading control. Secondary anti‐mouse and anti‐rabbit antibodies were used at 1:10,000 (Jackson ImmunoResearch Europe).