Dylight 554 phalloidin

For imaging, 10⁴ cells/well were cultured on a Falcon^® 96-well Black/Clear Flat Bottom TC-treated Imaging Microplate (Corning, New York, NY, USA), following the procedure described above. On the day of staining, cells were washed twice with PBS solution, fixed for 10–15 min in freshly prepared 4% paraformaldehyde (Avantor Performance Materials Poland, Gliwice, Poland), and permeabilized in 0.25% Triton X-100 (Sigma-Aldrich, Saint-Louis, MO, USA) for 15 min at room temperature. After two washes in PBS solution, cells were blocked for 30 min in 1% bovine serum albumin (Sigma-Aldrich, Saint-Louis, MO, USA) solution in 0.1% PBS/Tween 20 at room temperature. Then, the cells were rinsed with PBS three times (5 min each) and counterstained with DAPI (1:1500; Cell-Signaling, Danvers, TX, USA) and DyLight™ 554 Phalloidin (1:100; Cell-Signaling, Danvers, MA, USA) in PBS solution for 15 min at room temperature. Cells were reviewed and photographed under an Olympus IX81 fluorescent microscope (Olympus, Warsaw, Poland) with CellSense software (Olympus, Warsaw, Poland).

AGS, KATO III, NCI-N87 [N87] and HGC-27 cells were cultured for 24 h in 1% FBS medium and then were trypsinized, resuspended in 1% FBS medium and seeded in 96 multi-well plates. Immunofluorescence experiments were performed as previously described immunolabeling the cells in humidified dark chamber for 2 h with DyLight 554 Phalloidin (Cell Signaling, Beverly, MA, USA). Nuclei were stained with DAPI for 10 min in the dark. After rinsing with PBS, images were acquired with ZOE fluorescent cell imager (Bio-Rad, Milan, Italy)^{42 (link)}.

For the fluorescent staining of F-actin, the blades were cut into small (~2 × 2 mm²) pieces, which were immediately treated for 20 min in the dark with 300 μM m-maleimidobenzoyl-N-hydroxysuccinimide ester in PEMS buffer (50 mM PIPES, 5 mM EGTA, 5 mM MgSO₄, 4% NaCl, pH 6.8) + 0.1% Triton X-100 (v/v) to stabilize actin filaments. Afterwards, the samples were fixed for 1 h in 4% (w/v) paraformaldehyde + 5% (v/v) dimethylsulfoxide (DMSO) + 0.1% (v/v) Triton X-100. DyLight 554-phalloidin (Cell Signaling Technology, Danvers, MA, USA) at 1:400 was added to the fixative for better preservation of F-actin. After 3 × 15 min rinses with PEMS, the samples were extracted with 5% (v/v) DMSO + 1% (v/v) Triton X-100 in PBS for 1 h and afterwards F-actin was stained with DyLight 554-phalloidin 1:40 in PBS + 0.1% (v/v) Triton X-100 for 2 h at 37 °C in the dark. The cell wall was then counterstained with 0.05% (w/v) Calcofluor White in PBS for 10 min. After a final rinse, the samples were mounted on microscope slides in a drop of antifade solution (glycerol + PBS (2:1, v/v) + 0.5% (w/v) p-phenylenediamine). The above fluorescent specimens were examined using a Zeiss Observer.Z1 inverted microscope, equipped with a LSM780 CLSM module and ZEN2011 black edition software. Digital micrographs were recorded following the instructions of the manufacturer.

At 24 h after seeding the cells in 96-well plates with blacked walls (Corning, Amsterdam, The Netherlands), the tested compounds were applied at various concentrations. After 48 h of treatment, the cells were gently washed with PBS, fixed with 4% paraformaldehyde (Avantor Performance Materials, Gliwice, Poland) in PBS (4 °C, 10 min), permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, Poznań, Poland) in PBS (4 °C, 15 min), labeled with DyLight™ 554 Phalloidin (#13054, Cell Signaling Technology, Warsaw, Poland) for 10 min and DAPI (Thermo Fisher Scientific, Warsaw, Poland) for 5 min, visualized using an Olympus IX81 fluorescence microscope equipped with a XC10 camera and analyzed with CellSens Dimension software (all Olympus Polska, Warsaw, Poland).

Cells were plated on coverslips in six well plates and allowed to adhere overnight. For cytoskeletal staining, cells were fixed in 0.25% Glutaraldehyde/2% Paraformaldehyde for 15 minutes and coverslips were incubated with a FITC-conjugated antibody to detect vimentin (Alexa Fluor® 488 Conjugate-Vimentin, Cell Signaling Technologies) for 30 minutes. Coverslips were washed by dipping in PBS, then incubated for 15 minutes with DyLight™ 554 Phalloidin (Cell Signaling Technologies) to detect actin. Coverslips were washed by dipping in PBS. After a final wash in water, coverslips were placed on slides with mounting media containing DAPI (Life Technologies).

VK2 E6/E7 cells were plated in 24-well plates with 2% gelatin-coated glasses (6 × 10⁴ cells/well). After 24 h of settlement, cells were treated with vehicle or PE N/MPLs (25 and 250 µg/mL) for 72 h. Immunofluorescence of cells fixed in 4% paraformaldehyde was performed as previously described by Ceccarelli et al. [77 (link)]. Cells were incubated for 1 h at RT with anti-Lamin B and anti-β-Catenin primary antibodies (Santa Cruz Biotechnology, Dallas, TX, USA), followed by a specific Alexa Fluor™ 488-conjugated secondary antibody (Thermo Fisher Scientific) for 30 min. Actin filaments were labelled with DyLight™ 554 Phalloidin (Cell Signalling Technology, Danvers, MA, USA) for 1 h at RT. Cell nuclei were stained with 1 μg/mL 4’, 6-diamido-2phenylindole dihydrochloride (DAPI, Sigma-Aldrich). Images were acquired with a fluorescence microscope (Olympus BX53, Center Valley, PA, USA) using a 40× magnification.