Ix81 zdc

Real-time video recordings of monocytes were performed with an inverted phase-contrast microscope (Olympus, IX81-ZDC, Suffolk, U.K.) housed in a humidified, 5 % CO₂ gassed, temperature-controlled (37 °C) chamber. For this, 3 × 10⁵ freshly isolated monocytes were applied in each well of an IBIDI slide in the presence or absence of TsSP (40 μg/ml), and randomly selected fields were recorded for 240 min. Pictures were taken every 3 min with an Olympus ColorView II camera (Olympus Nederland BV). Recordings were analyzed using CELL F trackIT software (Olympus Soft Imaging Solutions, Münster, Germany).

Cells grown on 4-well chamber slide were treated with 1 μM Fura-2/AM (Invitrogen) in imaging medium for 20 min at 37°C. After washing twice, the cells were incubated with Locke's solution (158.4 mm NaCl, 5.6 mm KCl, 1.2 mm MgCl₂, 2.2 mm CaCl₂, 5 mm HEPES, and 10 mm glucose, pH 7.4) and then stimulated with DC. Intracellular Ca²⁺-dependent changes in fluorescence were imaged using an IX81 ZDC (Olympus) inverted microscope equipped with a 40 × oil immersion lens, and the produced at a rate of one image/3s. For ratiometric imaging, cells were alternately excited at 340 nm (Fura-2-Ca²⁺ complex) and 380 nm (free Fura-2) and measured at an emission wavelength of 500 nm. Changes in the F340/F380 nm fluorescence ratio were determined from selected regions across the cytoplasm of a single cell; 10–20 cells were imaged per well, and 30–60 cells were quantified per condition.

Cytoplasmic calcium concentrations were determined as previously described^{29 (link)}. Briefly, HeLa cells were labeled for 30 min with 5 μM Fura-2 and incubated at 37 °C/5% CO₂ in calcium-free Locke’s solution containing 154 mM NaCl, 5.6 mM KCl, 3.2 mM MgCl₂, 5 mM HEPES, 10 mM glucose, and 0.2 mM EGTA (pH 7.4). After drug treatment, changes in fluorescence were monitored every 5 min using an IX81 ZDC microscope (Olympus) at an emission wavelength of 510 nm, with dual excitation at 340 and 380 nm. Images of 340/380 fluorescence ratios were generated and analyzed using the Xcellence software package (Olympus). To detect HSF1 localization, HeLa cells were transfected with an HSF1–GFP plasmid^{30 (link)} obtained from Addgene (Addgene plasmid #32538) using the Lipofectamine transfection reagent (Invitrogen). After transfection for 24 h, cells were treated with the indicated drugs and then analyzed using an IX81 ZDC (Olympus). To visualize mitochondrial structure, HeLa cells were stained for 20 min with 200 nM MitoTracker dye, treated with the drugs, and then analyzed under an LSM 780 confocal microscope (Zeiss).

Capillary formation assay was performed as previously described^{6 (link)}. Briefly, ECs and fibroblasts were seeded together (20,000 cells/well of each cell type) in a 24-well plate in complete EC medium. After 9 days of culture (medium renewal: every 48 h), the cells were washed twice using PBS and fixed in cold methanol for 10 min. Fixed cells were blocked by 30 min of incubation in PBS containing 3% BSA. Fibronectin was stained with a mouse monoclonal anti-FN antibody (IST-2, 1:200 in PBS + 3% BSA, overnight at 4 °C). KLK12 was stained with a sheep polyclonal anti-KLK12 antibody (AF3095, 1:100 in PBS + 3% BSA, overnight at 4 °C). After three washes with PBS (5 min at RT), a secondary fluorescent antibody was applied (goat polyclonal anti-mouse IgG antibody - Alexa Fluor®-488 or donkey polyclonal anti-sheep IgG – NorthernLights-493, 1:1000 in PBS + 3% BSA, 1 h at RT). PECAM/CD31 was stained using an anti-CD31 antibody conjugated to phycoerythrin (FAB3567P, 1:100 in PBS + 3% BSA, overnight at 4 °C). Cell nuclei were stained with Hoechst 33342 reagent (diluted to 10 µg/ml in PBS) for 10 min at RT. Staining was visualized using a confocal microscope (Olympus IX81-ZDC, 20x and 40x lens). The resulting Z-stack images were superposed using Imaris Software, and maximum intensity projection images are presented (Bitplane, Zurich, Switzerland).

CFP and YFP images of HeLa cells and A549 cells were obtained by using an inverted microscope (IX81-ZDC; Olympus, Tokyo, Japan) equipped with a cooled CCD camera (Cool SNAP-K4; Roper Scientific), an illumination system (Spectra-X light engine; Lumencore, OR), an IX2-ZDC2 laser-based autofocusing system (Olympus), a MAC5000 controller for filter wheels and XY stage (Ludl Electronic Products, Hawthorne, NY), an incubator chamber system (Tokai Hit, Shizuoka, Japan) and a GM-4000 CO2 supplier (Tokai-Hit, Fujinomiya, Japan). The following filters were used for the dual emission imaging studies: FF01–438/24–25 (Semrock, Rochester, NY, USA), and FF01–475/28–25 (Semrock) excitation filter for CFP and YFP/GFP, a U-MREF glass reflector (Olympus) as a dichroic mirror, an FF01–483/32–25 emission filter (Semrock) for CFP and an FF01–542/27–25 emission filter (Semrock) for YFP. The microscope was controlled by MetaMorph software (Universal Imaging, West Chester, PA). The average fluorescence intensities of CFP and YFP in each cell were measured by manually delineating a region of interest at the cytoplasm with MetaMorph software.

For detection of Gαi1 and caveolin-1, cells were fixed with 4% paraformaldehyde followed by incubation with methanol at −20 °C for 10 min or 0.2% Triton X-100 at room temperature for 10 min. For detection of active β1-integrin, cells were incubated with medium including an anti-active β1-integrin antibody at 4 °C for 5 min. Subsequently, the cells were fixed with 4% paraformaldehyde followed by incubation with 0.2% Triton X-100 at room temperature for 10 min. The cells were blocked with 5% bovine serum albumin at 37 °C for 30 min, incubated with primary antibodies at 4 °C overnight, washed, and then incubated for 1 h with secondary antibodies (Alexa Fluor 488- or 594 goat anti-mouse, anti-rabbit or anti-rat IgG (Molecular Probes)). Fluorescent labelling was visualized using an inverted wide-field fluorescence microscope (IX81-ZDC, Olympus, Japan). Three-dimensional images (0.2 μm intervals) were obtained using an Olympus oil immersion objective lens (UPLSAPO 60XO/NA1.35) and computationally processed by three-dimensional deconvolution using Meta Morph software. For Figs 1i, 3b,d and 4a,b, images were obtained using an inverted confocal fluorescence microscope (Leica Microsystems, TCS SP8 with Hybrid Detector) and LAS-X software. Images were analysed using Image J software.