Fig 1: Meflufen induces DNA damage and apoptosis in cancer cells. (A) D492, D492HER2, and MDA-MB231 cells were seeded on a six well plate and incubated with either none, 1 µmol L-1 or 5 µmol L-1 melflufen for 30 min and harvested 3 h post-treatment. Equally loaded protein lysates subjected for immunoblotting for H2AX/ yH2Ax One representative blot of three is shown. (B) Quantification of protein bands of three independent experiments performed in 4A. Actin was used as loading control. Quantification was perfomed by FIJI. Shown are means ± standard deviation (n = 3). (C) D492, D492HER2, and MDA-MB231 cells were incubated with 1 µmol L-1 of melflufen for 30 min and harvested on indicated timepoints and subjected for immunoblotting for H2AX/ yH2Ax. One representative blot of 3 is shown. (D) Quantification of protein bands of three independent experiments performed in 4C. Actin was used as loading control. Quantification was perfomed by FIJI. Shown are means ± standard deviation (n = 3). (E) D492, D492HER2, and MDA-MB231 cells were cultured in monolayer and 24 h after treatment with none, 1 µmol L-1 or 5 µmol L-1 melflufen or melphalan, cells were fixed and stained for yH2Ax (red). IF analysis reveals that melflufen increases DNA damage in a dose dependent manner. Melphalan shows only a minor increase in staining at the concentrations used. Scalebar = 30 µm. (F) For immunostaining of D3 cultures, colonies of D492, D492HER2 and MDA-MB231 were incubated with either 10 µmol L-1 melflfufen or melphalan for 2h, fixed 72h post-treatment and stained for yH2Ax (red) and DAPI (blue). Scalebar = 100 µm (G) D492HER2 and MDA-MB231 cells were incubated with increasing amounts of melflufen for 24 h, harvested in RIPA buffer and subjected to immunoblotting for cleaved caspase-3. One representative blot of 3 is shown. (H) Quantification of protein bands of 3 independent experiments performed in 4G for caspase-3. Actin was used as loading control. Quantification was perfomed by FIJI. Shown are means ± standard deviation (n = 3)