4 6 diamidine 2 phenylindole dihydrochloride dapi

One day before infection, SH-SY5Y cells were plated on 22 × 22 mm square glass coverslips (Menzel-Glaser) in a 6-well plate (Costar, Corning, NY, USA) and incubated at 37 °C in a humidified 5% CO₂ atmosphere. The overnight B. pseudomallei culture was subjected to infection by the SH-SY5Y cells at an MOI of 20. After killing the extracellular bacteria as previously described, actin tail formation was observed at 6 h post-infection. The infected cells were washed with PBS twice and then fixed with 4% (v/v) paraformaldehyde in PBS at room temperature overnight. The fixed cells were washed with PBS before permeabilization with 0.5% (v/v) Triton X-100 in PBS. After 30 min of incubation, 1% (w/v) bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA) in PBS was added and incubated for 30 min at room temperature. Subsequently, bacteria were stained using a mouse monoclonal anti-B. pseudomallei lipopolysaccharide antibody (Camlab, Cambridge, United Kingdom) followed by Alexa Fluor488-conjugated anti-mouse Immunoglobulin (Molecular Probes, Eugene, OR, USA). Actin filaments and DNA were stained using Alexa Fluor555-conjugated phalloidin (Molecular Probes) and 4′,6′diamidine-2′-phenylindole dihydrochloride (DAPI; Molecular Probes), respectively. Actin-tail formation was examined in 100 fields by confocal laser scanning microscopy (LSM 700; Carl Zeiss, Jena, Germany).

l-Ala-γ-D-Glu-meso-diaminopimelic acid (TriDAP), muramyl dipeptide (MDP) and polyinosinic-polycytidylic acid (polyI:C) were purchased from Invivogen (San Diego, CA, USA). Macrophage-activating lipopeptide of 2 kDa (MALP-2) was synthesized and purified as described before [19 (link)]. Lipopolysaccharide (LPS) and isolectin B4 were from Sigma-Aldrich (Seelze, Germany), 4',6-Diamidine-2'-phenylindole dihydrochloride (DAPI) from Life Technologies^™ (Darmstadt, Germany), 4% formalin (Roti^®-Histofix) from Carl Roth (Karlsruhe, Germany). Rat anti-mouse CD31 and CD144 antibodies were obtained from BD Biosciences (San Jose, CA), Dynabeads sheep anti-rat IgG was purchased from Invitrogen (Carlsbad, CA, USA). VectaMount permanent mounting medium was used from Vector Laboratories (Burlingame, CA, USA).

HeLa and SiHa cells (10,000 cells/mL), transfected with shTCONS_00026907 (shRNA and NC), were cultured on a coverslip. Briefly, the treated HeLa and SiHa cells were fixed with 3.7% paraformaldehyde in PBS for 20 min at room temperature. 0.2% Triton X‐100 solutions in PBS were used to permeabilize cells for 10 min at room temperature. Then, cells were incubated with the primary antibody of p‐ELK1 (1:100, Santa Cruz Biotechnology, USA) overnight at 4°C. After being washed, the fluorescence‐conjugated secondary antibody (goat‐anti‐rabbit‐Alexa 594‐conjugated antibodies, Life Technologies, USA) was used to incubate the treated coverslips for 1 h at room temperature. The treated coverslips were then incubated with 4′,6‐Diamidine‐2′‐phenylindole dihydrochloride (DAPI; Life Technologies, USA) for 10 min at room temperature. The images were obtained using a fluorescence microscope (Olympus, USA).

NRCs were cultured on coverslips coated with laminin (BD Sciences). After adenoviral infection or siRNA transfection, serum starvation and stimulation with Ang II/PE, cells were fixed in 4% paraformaldehyde at RT, washed with PBS, permeabilized with 0.5% Triton X-100, and blocked with a blocking solution (1% BSA, 0.1% cold water fish skin gelatin, 1% Tween-20 in PBS) for 1 h. Cells were then labeled using an antibody against α-actinin (Sigma-Aldrich) and a secondary antibody (AlexaFluor 568, Life Technologies) to visualize cardiomyocytes. 4′, 6-diamidine-2′-phenylindole dihydrochloride (DAPI) was used to visualize the nucleus (300 nM, 5 min, RT; Life Technologies). For detecting the translocation of NFAT, cardiomyocytes infected with NFATc1-GFP, were stimulated with AngII/PE (1 μM/50 μM) for 15 min and processed for further fluorescence recordings.
Fluorescence microscopy was performed using a Zeiss Axiovert 135 microscope with a 63x oil immersion objective (Zeiss Plan Neofluar). Quantification of NFATc1 nuclear translocation was determined by measuring the mean grey value of NFATc1-GFP in the nucleus and in the cytoplasm using Image J software. The quotient of nucleus to cytoplasm was used to compare the localization of NFATc1. Cell areas were determined using Image J software.