Supplementary fig 1. PRIMA-1METinduces apoptosis in HCT116 p53-/- cells expressing p63 or p73 mutants corresponding to known hot spot mutations in p53. HCT116 p53-/- cells were transiently transfected with TAp63-R204W, TAp63-R304W, TAp73-R193H or TAp73-R193H. After treatment with PRIMA-1MET for 24 hours cells were assayed by FACS for apoptosis using Annexin V and propidium iodide staining.
Supplementary fig 2.PML/p63 double immunofluorescence staining of Saos-2-Tet-TAp63-R304W cells treated with 40 μM PRIMA-1MET for 18 hours. PRIMA-1MET induces redistribution of mutant p63 to PML-NBs (middle panel), and redistribution of mutant p63 and PML to nucleoli (lower panel).
Supplementary table 1.Cell cycle distribution of PRIMA-1MET-treated mutant TAp63gamma-expressing Saos-2 cells in the presence or absence of doxycycline, according to FACS-PI.
Supplementary Matherials and methods
Generation of TAp73α, TAp73βand TAp63γmutants
The TAp73α and TAp73β R193H mutants were generated by the QuickChange® XL site-directed mutagenesis kit (Stratagene, La Jolla, CA) using the mutagenesis primer 5’-CGTCGTGAAACACTGCCCCAACCACG-3’. TAp63γ R204W and R304W mutants were generated by GeneArt, Germany.
Apoptosis assay
HCT116 p53-/- cells were transfected using lipofectamine2000 according to the manufacturer’s instructions. Briefly, cells were plated in 6-well plates and transfected at 80% confluency. Twenty-four hours after transfection cells were re-plated in 12- well plates at low density, around 30% confluency, and allowed to reattach. Cells were treated with PRIMA-1MET and assayed by flow cytometry for apoptosis using Annexin V and PI staining (BD Biosciences, San Diego, CA).
Immunofluorescence staining
Cells were placed on cover slips in 6-well plates at a density of 1x104 cells/cm2 over night and incubated in the presence of compounds. Cells were fixed with 4% formaldehyde and permeabilised with 0.2% Triton-X. Staining was performed as described (Rökaeus et al., 2007). Antibodies were diluted in blocking buffer (2% bovine serum albumin, 0.2% Tween-20, 10% glycerol in phosphate-buffered saline (PBS)). Cover slips were mounted using Vectashield hardset mounting medium with DAPI (Vector Laboratories, Burlingame, CA). Images were collected using a Zeiss Axioplan 2 microscope, equipped with an AxioCam HRm camera. The software used was Axio Vision 4.7. For quantitative data, a minimum of 500 cells were analyzed.
Cell density information
Cells were plated, allowed to grow overnight and treated with PRIMA-1MET the following day. These were the amount of cells used in the different assays:
WST-1 growth suppression assay:H1299cells were grown in 96-well plates at a density of1 000 cells per well per 100 µl. Saos-2cells were grown in 96-well plates at a density of 3 000 cells per well per 100 µl.Cell cycle assay: Cells were grown in 12-well plates; H1299: 8x103 cells/cm2; Saos-2: 1x104 cells/cm2.Caspase activity assay:H1299 cells were placed in 12-well plates at an initial density of 8x103 cells/cm2.Keratine 14 staining:Cells were plated in 12-well plates at a density of 1.1 x 103 cells/cm2.Real Time RT-PCR: H1299 or Saos-2 cells were plated in 6-well plates at a density of 8x103or 1x104 cells/cm2.TransAm assay for DNA binding: Cells were grownat a density of 1.6x104 cells/cm2in 10-cm plates.Western blotting:Cells were grown (H1299: 8x103 cells/cm2 in 12-well plates; Saos-2: 1x104 cells/cm2 in 6-well plates)overnight followed by PRIMA-1MET-treatment or temperature shift.
Transfection
Cells were plated at a density of 1.1 x 103 or 1.6x104 cells/cm2in 12-well or 10-cm plates over night. Cells were transfected with 0.5 to 1μg DNA per well in 12-well plate or 1.2 and 3 g in 10-cm plates by using lipofectamine 2000 (invitrogen) according to manufacturer’s protocol.