Journal: International Journal of Molecular Sciences

Article Title: Albumin/Mitotane Interaction Affects Drug Activity in Adrenocortical Carcinoma Cells: Smoke and Mirrors on Mitotane Effect with Possible Implications for Patients’ Management

doi: 10.3390/ijms242316701

Figure Lengend Snippet: ( A ) Cytotoxic effect of mitotane and its metabolites in H295R cell model. Apoptosis analysis. H295R cells (1.5 × 10 5 cells/well) were seeded in 24-well plates in complete medium. After 24 h incubation with the appropriate concentration of each drug in study and with or without additive substance, cells were detached and decorated with FITC-Annexin V antibody. FACS analysis was performed with cell sorter/FACS facility. ( B ) Western blot analysis for the detection of apoptosis-related proteins. H295R cells were cultured for 24 h with mitotane 30 μM alone (blue) and with the addition of BSA 100 μM (red) or NuSerum 2.5% (green). A 30 μg volume of protein lysate from each condition in biological replicate was evaluated for apoptosis-related proteins by decorating with cleaved-PARP (89 kDa) and phospho-HSP27 (27 kDa) antibodies. ( C ) Cytotoxic effect of mitotane in NCI-SW-13 cell model. Concentration–response curve of drug-induced inhibition of cell viability in SW-13 cell model. SW-13 cells were cultured for 24 h with a range of mitotane concentrations (0–250 µM). This standard condition was “perturbed” by adding NuSerum 2.5% or the physiological condition (FBS 10%). Technical quadruplicates were performed for each drug condition and the experiments were replicated at least 2 times.

Article Snippet: Filters were incubated overnight at 4 °C with 1:1000 primary antibodies: cleaved PARP (Cell Signaling Technology (CST), #5625, Danvers, MA, USA); phospho-HSP27 (CST, #2405); β-actin (Sigma-Aldrich, A5316, St. Louis, MO, USA).

Techniques: Incubation, Concentration Assay, Western Blot, Cell Culture, Inhibition