Tunel assay

A total of 300 islets from each condition were harvested, washed, and snap-frozen in optimal cutting temperature compound (Tissue O.C.T. Labonord, Templemars, France) and then sliced in 12 μm sections. Apoptotic cells were stained using a Terminal deoxynucleotidyl transferase dUTP Nick End Labeling (TUNEL) assay (Millipore, Molsheim, France) and observed by florescence microscopy.
Hypoxic staining was performed using a Hypoxyprobe-1 Kit (HPI Inc. Burlington, MA, USA). Solid pimonidazole HCl was added in the islet cell cultures (150 μM). After 24 h culture, islets were fixed with 2% paraformaldehyde (PFA) for 10 min and then incubated with primary and secondary antibodies. The antibodies used in this study include anti-mouse immunoglobulin (IgG₁, 1 : 50 dilution; HPI, Inc.) and Alexa Fluor 488 goat anti-mouse IgG (dilution 1 : 200, Life technologies, Carlsbad, CA, USA).

Hepatic cell death was detected by using the in situ terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay (Millipore) according to the manufacturer's instructions. TUNEL‐positive nuclei were visualized by green FITC fluorescence.

TUNEL staining was performed as previously described [23 (link)]. Briefly, a TUNEL assay (Millipore, USA) was used according to the manufacturer's instructions. A fluorescence microscope (Olympus DX51) was used to evaluate apoptotic cells. Image-Pro Plus 6.0 was used to quantify all the images.

siSema6A, and siScr/BRAF^V600E cells, 24 hours post-transfection were plated onto fibronectin (FN)-coated dishes (20 μg/ml, SIGMA-Aldrich), or culture dishes or poly-l lysine coated slides and 24 hours later analyzed by immunofluorescence. The cells incubated with Phalloidin-Tetramethylrhodamine B isothiocyanate (TRITC) were counterstained with Hoechst to highlight nuclei (SIGMA-Aldrich). For TUNEL assay, siSema6A, and siScr/BRAF^V600E cells, 24 hours post-transfection were plated onto FN-coated or poly-l lysine coated slides, as above described. 48 hours post-transfection TUNEL assay was performed following manufacturer's instructions (Millipore). Microscope OLYMPUS BX53 was used to evaluate fluorescence and Tunel. Scale bars 20 μm. NRAS/Sema6A cells chemoinvasion was assessed as described [49 (link)] by overexpression of pUSENMC-Sema6A expression vector, kindly provided by Silvia Prislei (University Cattolica, Rome) [50 (link)]. Chemoinvasion or chemotaxis assays after depletion of Sema6A in BRAF and NRAS mutant cells, were performed as previously described [49 (link)]. Each assay was carried out in quadruplicate and repeated at least three times.

DNA fragmentation was evaluated using an ApopTag1 (Qbiogene) kit. In this method, the enzyme terminal deoxynucleotidyl transferase labels the 3-OH ends of DNA generated during apoptosis with biotinylated nucleotides. Immunoperoxidase staining was used to detect these fragments. The apoptosis detection kit enables distinction of apoptosis from necrosis by specifically detecting the DNA cleavage and chromatin condensation associated with apoptosis. Human melanoma cells were cultured on 8-well slides (20,000 cells per slide, Roth Germany) overnight for attachment. Then, the cells were treated with the investigated compounds at concentrations of 6 and 12.5 µM for 24 h. After treatment, the cells were fixed with 4% formalin in PBS for 10 min and permeabilized with 0.2% Triton X-100 in PBS for 5 min at room temperature. TUNEL assay was carried out according the manufacturer’s (Millipore) instructions. Nuclei were stained with hematoxylin. Then samples were mounted by DPX (Thermo Fisher Scientific, Germany) on glass slides. Cells with stained nuclei were investigated by counting 100 cells in 3 randomly selected fields. The analyses were performed by two independent investigators. Samples were evaluated with a BX51 upright light microscope (Olympus, Japan).