Af6000

For co-culture experiments images were taken every hour at different magnifications with an inverted fluorescence microscope (Leica AF6000). Cancer cell elongation was quantified manually by scoring individual cells using Image J “Cell Counter” plugin on taken images at 48 h co-culture. Elongated cell were defined as single cell with a minimal length-width rapport of 2:1, as well as at least one sharp-ending extremity. Cancer cells motility was quantified based on single cell track follow on 24 h microscope movies using Image J “Manual Tracking” and “Chemotaxis Tool” plugins.
Imaging of 2D and 3D spheroids assay were performed with 5× and 40× objectives with Leica AF6000 inverted fluorescence microscope. Invasion was quantified using Photoshop CS6 (Adobe) by calculating the invaded area on the 4 days image, normalized on initial spheroid size.
Western Blot images were quantified for protein level using Image J “Gel Analyze Tool” plugin.
For immunostaining images were taken using a DeltaVision Elite Microscope (GE Healthcare) with 63× and 100× objectives, followed by deconvolution treatment.
For immunohistochemistry images, slides were scanned using a NanoZoomer 2.0 HT (Hamamatsu) and images were extracted using NDP.view2 analysis software (Hamamatsu).

To examine cell migration, live-cell imaging was performed on a Leica AF6000 inverted microscope at 37 °C under 5% CO₂. H1299 cells were seeded in six-well plates and grown to 100% confluence. A ‘wound’ was created using pipette tips. Cells were washed with 1 × PBS and maintained in DMEM with 1% FBS. The cell images at exact same place were recorded every 30 min using a time-lapse microscopy (Leica AF6000).

Hearts fixed in 4% paraformaldehyde were embedded in paraffin and then cut into 5 μm sections. Sections were incubated with an anti-HMGB1 rat polyclonal primary antibody (1:200 dilution, 60 min at room temperature; Abcam, USA) and a secondary antibody (1:100 dilution, 60 min at room temperature; Zhongshan Biotechnology, China). The sections were incubated in HRP-streptavidin (1:100; Zhongshan Biotechnology, China) for 30 min at 37°C, and the color reaction was visualized with diaminobenzidine (DAB; ZSGB-BIO, Beijing, China). For each section, five fields were randomly selected from the infarcted area of the left ventricle and photographed with a digital camera (AF6000, Leica, Germany). The mean density of HMGB1 expression in the myocardium was analyzed using Image J.