2.7. Gal‐1‐induced T‐cell apoptosis assay

A375 control and A375_GAL1sh cells plated on 24‐well plate were treated with DMSO alone or vemurafenib (2.5 µm) for 24 h. Jurkat T cells were stimulated for 24 h using human T‐activator CD3/CD28 Dynabeads (Thermo Fisher Scientific), and 1 × 10⁵ of activated cells were mixed with pretreated A375 and A375_GAL1sh cells. After 24 h, apoptosis of Jurkat T cells was assessed in cocultures by flow cytometry. Cells were stained with APC‐CD7 antibody to discriminate T cells from A375 cells (T cells: CD7‐positive; A375 cells: CD7‐negative) and subsequently with Annexin V–fluorescein isothiocyanate (FITC) to evaluate apoptosis. Annexin V‐FITC staining was performed according to the manufacturer’s instructions (Annexin V‐FITC Apoptosis Detection Kit I, BD Biosciences). Apoptotic cells were evaluated in the CD7‐positive population using BD FACSCanto flow cytometer (BD Biosciences) and analyzed by flowjo v.10 software (BD Bioscience).