Alexa 633 phalloidin

COS-7 cells were cultured on gelatin-coated coverslips in 12-well plates. Transfection was performed when the cell confluence reached 40%. COS-7 cells were individually transfected with mCherry or mCherry-tagged Homer1 (WT, R3E, and R3E-ENAH) by ViaFect Transfection Reagent (Promega). Briefly, 1 μg DNA and 3 μL transfection reagent were mixed in 100 μL Opti-MEM media (GIBCO) for 20 min at room temperature before the mixture was added into each well. Seventeen hours after transfection, cells were fixed with 4% (vol/vol) PFA together with 4% (wt/vol) sucrose in PBS (pH 7.5).
For F-actin staining, fixed cells were first permeabilized with 0.2% Triton X-100 in PBS (pH 7.5) for 20 min, and then blocked by blocking buffer containing 5% (wt/vol) BSA in PBS (pH 7.5) for 2 hr at room temperature. After blocking, cells were stained with Alexa-633 phalloidin (Thermo Fisher, 1:1000 in blocking buffer) for 1 hr at room temperature. Mounted cells were imaged at Nikon Ni-U upright fluorescence with a 40× lens. The percentage of cells with mCherry signal in lamellipodia were quantified by ImageJ. Statistical data was plotted from three independent batches of cells with >300 cells for each batch in a blinded manner.

Samples for fluorescent gelatin degradation and 3D culture experiments were fixed and prepared for immunofluorescence according to their respective protocols (see below). MT1-MMP primary antibody (AB6004) was detected using anti-rabbit IgG Alexa594 or Alexa488 for Oregon green-488 gelatin degradation or 3D culture experiments respectively. F-actin was stained with Alexa633 phalloidin (1:100, Thermo Fisher) and nuclei with 4′,6-diamidino-2-phenylindole (1 μg/ml, BioShop Canada). Samples were imaged using a Nikon A1R+ confocal microscope (1.2 au) with a 20× dry or 60× oil-immersion lens and presented using NIS Elements software.

5x10⁴ LLC1 cells were plated in 24 well plates on glass cover slips in DMEM or embedded into 0.5 mg/ml lr-ECM/DMEM mixture under 2D or 3D cell culture conditions, respectively. Following 48 h of growth, cells were washed twice with PBS and fixed for 10 min. with 4 % PFA (Carl ROTH, Germany) solution in PBS at room temperature. Cell permeabilization was performed with ice-cold 0,1 % Triton X-100 in PBS for 10 min. Staining was accomplished with Alexa®633 Phalloidin (ThermoFisher Scientific, USA) in PBS containing 1 % BSA for 30 min and 5 μg/ml Dapi (Sigma, USA) in PBS for 3 min at room temperature. All staining steps were followed by 3 wash steps in PBS for 5 min at room temperature. Finally, slides were mounted with Roti®-MountFluorCare mounting media (Carl ROTH, Germany). Images were obtained using Zeiss LSM 7 Duo Live confocal microscope (Zeiss, Germany) and 40x/1.3 immersion objective and excitation wavelengths of 405 nm and 633 nm.

For immunocytochemistry, the cells were treated with 4% paraformaldehyde (PFA) for 10 min for fixation, permeabilized with 0.2% Triton X-100 in PBST for 30 min and blocked with 5% goat serum (Jackson Laboratory, Bar Harbor, ME, USA) in PBS for 1 h. After that, the cells were incubated with rabbit anti-DsRed2 antibody (1:400, Clontech, Mountain View, CA, USA) or rabbit anti-Flag antibody (1:400, Sigma-Aldrich, St. Louise, MO, USA) diluted in 5% goat serum overnight at 4 °C. Then, the cells were incubated with Alexa 488- or 568-conjugated secondary antibodies (1:400, Thermo Scientific, Waltham, CA, USA) for 1 h at room temperature, and Alexa 633-phalloidin (1:800, Thermo Scientific, Waltham, CA, USA) was used to counterstain the actin filaments. The samples were mounted onto slides with Vectashield (Vector lab, Burlingame, CA, USA), and images were analyzed using a Zeiss LSM 700.

The following antibodies and reagents were used for the analyses: the rabbit antibody against Coronin 1B (Sigma-Aldrich); mouse antibody against CD144 (VE-cadherin) (eBioscience), rabbit antibody against integrin-linked kinase (ILK) (Cell Signaling Technology) and rabbit antibody against α-parvin (Cell Signaling Technology). For secondary detection, species-specific Alexa Fluor-coupled secondary antibodies (Invitrogen) were used. Filamentous actin (F-actin) was visualized with Phalloidin Alexa-633 (Invitrogen). HUVEC cells were treated with thrombin (Sigma-Aldrich) and Y-27632 (Merck Millipore).