Sc 6243

The ability of the newly synthesised compound RM37 to dissociate the native MDM2–p53 complex was assessed by a quantitative immuno-enzymatic assay, as previously reported [19 (link)]. Briefly, lysate samples of GBM cells were incubated for 10 min at room temperature with the solvent DMSO (control) (Sigma-Aldrich, Milan, Italy) or with increasing concentrations of the compound RM37 (from 1 nM to 50 µM) before being transferred to the wells coated with the antibody anti-MDM2 (sc-965, Santa Cruz Biotechnology, 1550 in 0.05% poly-L-ornithine) (Sigma-Aldrich, Milan, Italy). Following washings to remove unbound MDM2 and BSA treatment to block nonspecific sites, each sample was incubated for 90 min with the anti-p53 antibody (sc-6243, Santa Cruz Biotechnology, 15250 in 5% milk). The levels of the MDM2–p53 complex were quantified using an HRP-conjugated antibody and the TMB substrate.

Protein levels was determined as previously described [36 (link)] with antibodies against p53 (SC-6243, 1:500, Santa Cruz), p-p53ser15 (9284S 1:500, Cell Signal), p21 (SC-6246, 1:1000, Santa Cruz), BAX (SC-493, 1:500, Santa Cruz), c-PARP (9542 S 1:500, Cell Signal), and β-actin (2228, 1:20000, Sigma Aldrich).

Organoids were treated with 10 µM Nutlin-3a to activate p53 pathway 24 h prior to the harvesting. Samples were lysed using RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% SDS, 0.5% Na-Deoxycholate, 1% NP-40) containing Complete protease inhibitors (Roche). Protein content was quantified using standard Bradford assay (BioRad) and equal amounts of protein (a′ 20 µg) were run on gradient polyacrylamide gel (4–15%; BioRad) and transferred to PVDF membranes (Millipore). Membranes were blocked and probed with antibodies directed against p53 (sc-6243, 1:250, Santa Cruz Biotechnology) and GAPDH (LN2100751, 1:1000, Labned). Uncropped versions of the western blots are provided in the Supplementary Fig. 6. The results were confirmed twice.

Embryos (n = 38, 3 replicates) were fixed in 3.7% paraformaldehyde for 20 min at room temperature, permeabilized with PBS/PVA containing 0.5% Triton X-100 at 37 °C for 1 h, and then incubated in PBS/PVA containing 1.0% bovine serum albumin at 37 °C for 1 h. Subsequently, the embryos were incubated overnight at 4 °C with anti-LC3 (ab58610, 1:100; Abcam, Cambridge, UK), anti-cytochrome C (ab110325, 1:100; Abcam), anti-p53 (sc6243, 1:100; Santa Cruz Biotech, CA, USA) and anti-OCT4 (sc8628, 1:100; Santa Cruz Biotech) antibodies. To check the fluorescence signal of 5mC, blastocysts were denatured with 1 N HCl at room temperature for 30 min and neutralized with 0.1 M Tris-HCl, pH 8.0 for 15 min. Subsequently, blastocysts were incubated in PBS containing 1% BSA, and then incubated overnight at 4 °C with 5mC antibody (ab10805, 1:100, Abcam, Cambridge, UK). After washing three times with PBS/PVA, the oocytes and embryos were incubated at 37 °C for 1 h with either goat anti-rabbit IgG (A11011, 1:200, Invitrogen) or rabbit anti-goat IgG (A11079, 1:200, Invitrogen), anti-mouse IgG (A21202, 1:200, Invitrogen). The oocytes and embryos were then stained with Hoechst 33342 for 5 min, washed three times with PBS/PVA, mounted onto slides, and examined using a confocal microscope (Zeiss LSM 710 META, Jena, Germany). Images were processed using Zen software (version 8.0, Zeiss, Jena, Germany).

Four proteins, p53, p16, p27 and c-erbB2, which have been demonstrated to be independent prognostic factors for non-small cell lung cancer (NSCLC) (13 (link)–16 (link)), were selected for the differential diagnostic analysis of MPLC and IPM. IHC staining was performed using serial sections obtained from the same paraffin-embedded blocks. The specimens were stained with hematoxylin and eosin in order to confirm the histological diagnosis. IHC staining was performed using the streptavidin-biotin-peroxidase complex method. For the antigen retrieval, sections were briefly immersed in a citrate buffer (0.01 mol/l citric acid; pH 6.0) and then incubated for 25-min intervals at 100°C in a microwave oven. Next, the sections were incubated with a monoclonal mouse anti-p53 antibody (dilution, 1:100; sc-6243, Santa Cruz Biotechnology, Dallas, TX, USA), a polyclonal rabbit anti-p16 antibody (dilution, 1:200; ab54210, Abcam, Cambridge, MA, USA), a monoclonal mouse anti-p27 antibody (dilution, 1:250; ab32034 Abcam) and a monoclonal mouse anti-c-erbB2 antibody (dilution, 1:100; ab2428, Abcam) overnight in a cold room using a labeled streptavidin biotin kit (Dako LSAB kit; Dako, Carpinteria, CA, USA). The antibodies were diluted in phosphate-buffered saline containing 2% bovine serum albumin.