Nutlin 3

Cells from frozen cultures were seeded in 25 cm2-culture flasks. Before testing the compound, the cells must be reseeded at least once. To test the sensitivity to the compound treatment, the lung cancer cells in the exponential growth phase were seeded in wells of a 24-well plate at 60,000 cells per well. After 24 h, the culture medium was replaced with a fresh one containing the compound in the following concentrations: Nutlin-3 (Nutlin3 (±), Cayman Chemical) at concentrations of 34 μM, 17 μM, 8.5 μM, 4.25 μM, 2.2 μM and 0 μM (control).
Each concentration point was represented in triplicates. 48 h after the addition of the compound the proportion of viable cells was determined by a viability test with resazurin.

For in vitro treatments with Ibrutinib (PCI-32765) (Selleckchem, Houston, TX), used either alone or in combination with Nutlin-3 (Cayman Chemicals, Ann Arbor, MI), cells were seeded at a density of 1x10⁶ cells/mL. In selected experiments, Ibrutinib was assessed also in combination with the RG7112 MDM2 inhibitor (Selleckchem). At different time points after treatment, cell viability was examined by Trypan blue dye exclusion and MTT (3-(4, 5-dimethilthiazol-2yl)-2, 5-diphenyl tetrazolium bromide) colorimetric assay (Roche Diagnostics Corporation, Indianapolis, IN) for data confirmation, as previously described [41 (link), 42 (link)]. In order to investigate the concentration required to induce death in 50% of cells respect to control, IC₅₀ values were calculated from dose-response curves constructed by plotting cell survival (%) versus drug concentration. The cell cycle profile was analyzed by 5-bromodeoxyuridine (BrdU) incorporation assessed by flow cytometry, as previously described [43 (link)]. Levels of apoptosis were quantified by Annexin V-FITC/propidium iodide (PI) staining (Immunotech). To avoid non-specific fluorescence from dead cells, live cells were gated tightly using forward and side scatter, as described [44 (link)].

Transfections with pcDNA3-HSP70-HA (Invitrogen, Carlsbad, CA, USA) and HSP70 shRNA (Santacruz biotechnology, Dallas, TX, USA) were performed using Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. After transfection, the cells were treated with 200 nM α-MSH) for 24 h. Then, the cells were irradiated with RF, and maintained for 48 h.
The p53 inhibitor (Nutlin-3; Cayman chemical, Ann arbor, MI, USA) of cultured cells was treated at 50 μM for 24 h. After 24 h, the cells were treated with 200 nM α-MSH for 24 h. Then, the HEMn cells were irradiated with RF and incubated for 48 h.

NGP cells were synchronized using serum starvation prior to nutlin-3 treatment. First, cells were seeded at low density for 48 h in complete medium. Then, cells were refreshed with serum-free medium for 24 h. Finally, the cells were treated with either 8 μM of nutlin-3 (Cayman Chemicals, 10004372, dissolved in ethanol) or vehicle. Cells were trypsinized (Gibco, 25300054) 24 h post treatment and harvested for single cell analysis, bulk RNA isolation and cell cycle analysis.