Immunofluorescence Assay for γH2AX and WGA in A375 Cells

For immunofluorescence experiments, A375 cells were seeded at a density of 5 × 10^{3 (link)} in 12-well cluster plates in DMEM supplemented with 1% FBS. After 24 h, cells were treated with compounds 1 or 2 (0.5 and 1 μM for 48 h). Cells were then washed with phosphate-buffered saline (PBS), fixed with ice-cold methanol for 5 min and permeabilized with Triton 0.2% in PBS for 10 min. Immunostaining of γH2AX was performed as previously described^{57 (link)}. For Wheat Germ Agglutinin (WGA) immunostaining, cells were labeled as previously described^{58 (link)}. Samples were visualized on a TSC SP5 confocal microscope (Leica Microsystems, Milan, Italy) installed on an inverted LEICA DMI 6000CS microscope, using PlanApo 40 × 1.25 NA objective or PlanApo 63 × 1.4 NA oil immersion objectives. Images were acquired using the LAS AF acquisition software (Leica Microsystems). Fluorescence intensity measurements were performed using the Quantitation Module of Volocity software (Perkin Elmer Life Science, Milan, Italy).