The STS cell lines, IB130 (pleiomorphic liposarcoma/ mutant TP53 exon 8, P278L), IB134 (uterine leiomyosarcoma/mutant TP53 exon 6, S215R), IB136 (soft-tissue leiomyosarcoma, null TP53), IB117 (myxofibrosarcoma, null TP53), IB138 (soft-tissue leiomyosarcoma/mutant TP53 exon 5, V143M), and IB139 (soft tissue leiomyosarcoma, wild-type TP53) used in this study have been derived from human surgical specimen of STS in the laboratory of Pr. Jean-Michel Coindre and Dr Frédéric Chibon (Institut Berognié, Bordeaux, France) and after having obtained patient consent. For all the cell lines, TP53 status was assessed by Sanger sequencing, array-comparative genomic hybridization and western blotting (protocols are available on request). Colon carcinoma cell lines used were HCT-116 (wild type TP53) and HT-29 (mutant TP53 exon 8, R273H) purchase from the NCI (http://discover.nci.nih.gov/cellminer/). All cell lines were cultured in complete RPMI 1640 (Sigma Life Technologies, Saint Louis, MO) with 10 % Fetal calf serum, Penicillin/Streptomycin 1 %, and Normocin 0.2 %.
PRIMA-1MET and Staurosporin were purchased from Santa Cruz Biotechnology INC (Heidelberg, Germany). PRIMA-1MET was stored at −20 °C and diluted in water. Chloroquine Diphosphate salt and N-acetyl-L-cysteine were purchased from Sigma Life Science (Saint Louis, MO).
Three thousand cells were seeded in 96-well plates for 24 hr and treated with a range of increasing concentrations of PRIMA-1MET for 24 hr to 96 hr. Methyl Thiazolyl Tétrazolium (MTT, Sigma Aldrich, St Quentin Fallavier, France) was applied for 3 hours before being dissolved in dimethylsufoxyde (final concentration: 0.5 mg/mL). Quantity of produced formazan was measured by spectrophotometry. Absorbance was measured at 570 nm with a reference at 630 nm. Analysis was done by using the KC4 software (Kinetical for Windows V.3.4) and IC50 was calculated with GraphPad Prism version 5.00 for Windows (GraphPad Software, La Jolla California USA, www.graphpad.com).
Fluorescent cell sorting analysis (FACS)
Apoptosis and cell cycle were evaluated using Fluorescent Activating Cell Sorting (FACS) analysis. For mitochondrial membrane depolarization studies, 3000 cells were seeded in 96-well plates for 24 hours and treated with a range of increasing concentrations of PRIMA-1MET for 72 hours, then incubated for 30 min with Tetramethylrhodamine Methyl Ester (TMRM). P-glycoprotein drug efflux pump was blocked using Verapamil (Sigma-Aldrich, St Quentin Fallavier, France). For activated caspases 3 and 7 detection, 5.105 cells were seeded in 6-wells plates for 24 hours, treated with increasing doses of PRIMA-1MET for 72 hours and 96 hours, respectively. Cells were harvested and exposed to FLICA 1X as described by the supplier (FAM-FLICA™ Kit, ImmunoChemistry Technologies, Bloomington, USA) for 1 hour. For apopotosis/necrosis assay, 1.106 cells were seeded in 6-wells plates for 72 hours, then treated and exposed to FITC-Annexin and propidium iodide (PI) according the manufacturer’s protocol (BD Biosciences, Erembodegem, Belgium). This allowed distinguishing annexin V positive cells in early apoptosis, versus annexin V and PI positives cells in late apoptosis or necrosis. For cell cycle analysis, 1.105 cells were seeded in 6-wells plates and after 24 hours cells were treated with PRIMA-1MET for 48 hours to 96 hours. Cells were then fixed and permeabilized in absolute ethanol with PBS over-night at −20 °C, then rinsed and incubated with RNase and Propidium Iodide (50 μg/mL) (Sigma Aldrich, St Quentin Fallavier, France). For ROS production assay, 6000 cells were seeded in 96-well plates for 24 hours and treated with PRIMA-1MET for 96 hours, then incubated for 30 min with 2',7'-Dichlorofluorescin diacetate (DCFDA) 20 μM (Abcam, Cambridge, MA, USA). We performed flow cytometry analysis using FL1 for TMRM, FAM-FLICA, Annexin-V and DCFDA, whereas FL2 was used for Propidium iodide. Flow cytometry (FACSCalibur; BD Biosciences, San Jose, USA) data were analyzed with FlowJo v.7.6.3. software.
The physical lysis « Ultimate freeze-thaw lysis for mammalian cells » protocol was used for non-phosphorylated protein extraction . For autophagy and phosphorylated protein, Radio-ImmunoPrecipitation Assay (RIPA) lysis buffer protocol  was used. Total proteins (30-60 μg) were separated by 10 or 12 % SDS-PAGE and transferred on Polyvinyl Difluoride (PVDF) membrane. The following antibodies were purchased from Cell Signaling Technology (Danvers, USA): anti-BAX (1:500), anti-PUMA (1:1000), anti-JNK (1:1000) and anti-phospho JNK monoclonal antibodies (1:1000). Anti-GAPDH (1:200) and anti-p53 (1:1000) monoclonal antibodies were purchased from Santa Cruz Biotechnology (Heidelberg, Germany). The anti-p21 monoclonal antibody (1:33) was purchased from Calbiochem (San Diego, USA). The anti-LC3IIB monoclonal antibody (1:1000) was purchased from Sigma Aldrich (Saint Louis, USA) and the anti-PARP monoclonal antibody (1:1000) was purchased from EnzoLifeSciences (Farmingdale, USA). Secondary antibodies anti-Mouse IgG and anti-Rabbit IgG (1:5000) were purchased from Ge Healthcare (Buckinghamshire, United Kingdom). Proteins were detected (Fusion Fx7, Fisher Bioblock Scientific, Waltham, MA, USA) by using enhanced chemiluminescent substrate for horseradish peroxidase (HRP) (Immobilon™ Western, Millipore Corporation). Each membrane was reused 3 times after desaturation in glycin (6.6 mol/L) buffer pH = 2 at 56 °C for 30 min. Semi-quantitative analysis was realized with ImageJ 1.45 s software (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/, 1997–2014).
Cells were seeded on coverslips and treated with PRIMA-1MET for 72 hours. Slides were then washed twice with PBS, fixed in formaldehyde 4 % and incubated with anti-LC3IIB monoclonal antibody (Sigma Aldrich, Saint Louis, USA) overnight, and then with a goat anti-rabbit Alexa fluor 488 antibody (Invitrogen, Paisley, United Kingdom). Slides were then counterstained by 4,6-diamidino-2-phenylindole (DAPI).