Las af acquisition software

NTera2/D1 cells were washed with PBS, fixed with ice-cold methanol for 15 min, and incubated with γH2A.X- or 53BP1-specific primary antibody for 1 h. Alternatively, they were washed with PBS, fixed with 4% paraformaldehyde in PBS for 20 min and permeabilized with 100 μg/ml digitonin (Life Technologies) in PBS for 15 min, and incubated with LC3B-specific primary antibody for 1 h. Cells were then washed three times with PBS, incubated for 30 min with appropriate Alexa Fluor 488-conjugated or Alexa Fluor 555-conjugated secondary antibodies and washed again three times in PBS. Nuclei were stained with 1.5 μM 4′,6-diamidino-2-phenylindole (DAPI; Sigma Aldrich) in PBS for 5 min. Coverslips were mounted in Fluorescence Mounting Medium (Dako, Milan, Italy). 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). At least five representative fields were acquired and analyzed for each sample.

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).