Texas red phalloidin

Ten microliters of 100% Matrigel (Sigma-Aldrich Chemie Gmbh, Munich, Germany, Corning art. 356230) was used to coat the bottom of each well of an eight-chamber slide. A suspension containing 15,000 cells/mL and 4% Matrigel was added on top of the first coating. Spheres were allowed to form for 6 days after which the media was replaced with media containing DMSO, afatinib, or crizotinib alone or in combination. The drug exposure was done for 72 h. Media was removed and each well was washed twice with 1× PBS. The slides were fixed in 4% buffered formaldehyde for 15 min and thereafter washed twice with 1× PBS, permeabilized with Triton X-100 for 1 min, and blocked with 5% horse serum for 1 h. Slides were then incubated for 1.5 h with Texas Red × Phalloidin (Thermo Fisher Scientific, Waltham, MA, USA). Slides were washed thrice with 1× PBS, mounted with Fluoroshield containing DAPI (Sigma-Aldrich Chemie Gmbh, Munich, Germany) and imaged using Zeiss AxioImager M2 microscope.

Cells seeded onto coverslips were grown to 30˗40% confluence and then transfected with plasmids or treated with CoCl₂ as described above. After 24˗72 h, cells were fixed with 3.7% formaldehyde for 15 min and permeabilized with 0.5% Triton X-100 for 15 min at room temperature. After blocking with 10% normal goat serum, coverslips were incubated with primary antibodies (1:200) or Texas Red™-phalloidin (for F-actin staining; Thermo Fisher) at 4°C overnight. After washing, coverslips were incubated with secondary antibodies conjugated with FITC or Cy3 (1:200; Bethyl) for 1 h at room temperature. Coverslips were incubated with mounting solution containing DAPI and observed under a fluorescence microscope (Zeiss).

The EF-treated and untreated cells were cultured on slips and rinsed twice in pre-warmed (37 °C) PBS. Then these cells were fixed in 4% paraformaldehyde for 20 minutes, permeabilized with 0.3% TritonX-100 for 20 minutes, blocked with goat serum for 1h, and incubated with primary antibodies overnight at 4 °C. For immunofluorescence microscopy, the secondary antibody was coupled to Alexa 488, and the nuclei or whole cell areas were stained with DAPI or cell membrane blue, respectively. Of F-actin labeling fixed cells were incubated with Texas Red phalloidin (1:250, T7471, Thermo Fisher Scientific) for 20 minutes in the dark. The expression of α-SMA, F-actin and DAPI were observed under Leica Confocal Microscope (Leica Microsystems, Wetzlar, Germany). The excitation wavelengths for α-SMA, F-actin, and DAPI were set at 488nm, 680nm, and 405nm respectively, while the corresponding emission wavelengths were 520nm, 720nm, and 450nm. The images were captured with an exposure time of 200ms and a gain of 1.5x. Eventually, the images were processed using Image J.

Antibodies that recognize ERK1/2, p-ERK1/2, RAF1, p-RAF1, AKT, p-AKT, MEK1/2, p-MEK1/2, GAPDH, COXIV, VDAC1, cleaved caspase-3, calnexin, PCNA, E-cadherin and vimentin were purchased from Cell Signaling Technology (Beverly, MA). Antibodies against β-actin, Myc, and Flag were obtained from Sigma-Aldrich (St Louis, MO). Antibodies that recognize cyclin D1 and CDK4 were purchased from Santa Cruz Biotechnology (Dallas, TX). ATAD3A antibody was purchased from Novus Biologicals (Abingdon, UK). CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) was obtained from Promega (Madison, WI). Texas-red phalloidin, D-luciferin bioluminescent substrate, and alamarBlue Cell Viability Reagent were purchased from ThermoFisher Scientific (Waltham, MA). The RAS inhibitor salirasib and the ERK1/2 inhibitor SCH772984 were obtained from SelleckChem (Houston, TX), and 4-nitroquinoline-1-oxide (4NQO) was purchased from Sigma-Aldrich (St. Louis, MO).

NRCMs (5 × 10⁵ cells/ml) were seeded in 24-well plates and routinely cultured. The cells were fixed with 4% paraformaldehyde and permeabilized with 1% Triton X-100 in PBS for 5 min. The cells were then washed with PBS and stained with Texas Red-Phalloidin (Thermo Fisher) for 30 min at room temperature. The nuclei were stained with 0.1 μg/ml DAPI for 5 min. The cells were visualized with a fluorescence microscope, and the cell surface area was measured by analyzing 30 NRCMs from at least five random fields with ImagePro Plus 6.0 Software (Media Cybernetics, Bethesda, MD, U.S.A.).

The chemicals used for our method were purchased as shown in Table 1. The antibodies were those against VE-cadherin 160840; Cayman, Ann Arbor, MI, USA, ICAM-1 3422R-100; BioVision, Milpitas, CA, USA and Ac-LDL Sigma-Aldrich, St Louis, MO, USA. Secondary antibodies were Alexa Fluor^® 488 dye and Alexa Fluor^® 594dye Thermo Fisher Scientific, West Columbia, SC, USA. Lectin Bandeiraea simplicifolia Griffonia simplicifolia BS1 was obtained from Santa Cruz, CA, USA and lectin Helix pomatia Alexa Fluor 488conjugateLife Technologies, Carlsbad CA, USA and Texas Red Phalloidin was purchased from Thermo Fisher Scientific, Grand Island, NY, USA. Eight-well arrays were from Applied Biophysics Albany, NY, USA.