Supplementary Figure 4A: Scheme of the lentiviral vectors, as integrated proviruses, that have

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1 Suppl. Figure 4 LeGO-iG2-Puro+-p53 SIN-LTR ψ RRE cppt SFFV human Wt-p53 IRES egfp2a PuroR_opt wpre SIN-LTR LeGO-G2-Puro+ SIN-LTR ψ RRE cppt SFFV egfp 2A PuroR_opt wpre SIN-LTR Supplementary Figure 4A: Scheme of the lentiviral vectors, as integrated proviruses, that have been used in this study (not drawn to scale). SIN-LTR, self-inactivating-long-terminal repeat; Ψ, packaging signal; RRE, rev-responsive element; cppt, central polypurine tract; SFFV, Spleen focus-forming virus enhancer/promoter; human Wt-p53, cdna coding for human wild-type p53; IRES, internal ribosome entry site of the Encephalo myocarditis virus; egfp (enhanced green fluorescent protein), a green fluorescent protein; 2A, self cleaving peptide of Porcine Teschovirus-1 (P2A); PuroR-opt, codon optimized cdna of puromycin resistance (PAC, puromycin N-acetyltransferase); wpre, Woodchuck hepatitis virus post-transcriptional regulatory element.

2 A) UKF-NB-3rNUTLIN10µM B) UKF-NB-3rNUTLIN10µM C) UKF-NB-3rNUTLIN10µMwtp53 D) UKF-NB-3rNUTLIN10µMwtp53 E) UKF-NB-3rNUTLIN10µMcontrol F) UKF-NB-3rNUTLIN10µMcontrol Supplementary Figure 4B: UKF-NB-3rNUTLIN10µM cells (A + B), UKF-NB-3rNutlin10µM cells transduced with LeGO-iG2-Puro+-p53 (UKF-NB-3rNUTLIN10µMwtp53) (C + D) and UKF-NB3rNutlin10µM cells transduced with LeGO-G2-Puro+ (UKF-NB-3rNUTLIN10µMcontrol) (E+ F) were

3 photographed by inverse light microscopy (A, C, E) and by fluorescence microscopy (B, D, F), using an IX71 fluorescence microscope (Olympus, Hamburg, Germany). Fluorescence dye egfp (enhanced green fluorescent protein) was detected at 484/507nm (excitation/emission). The nontransduced control cell line UKF-NB-3 r NUTLIN 10µM showed no fluorescence (B). In contrast, UKF- NB-3 r NUTLIN 10µMwtp53 and UKF-NB-3 r NUTLIN 10µMcontrol cells displayed high fluorescence due to successful transduction with LeGO-vectors (D, F).

4 p53 β-actin Supplementary Figure 4C: Western Blot was carried out with UKF-NB-3, UKF-NB-3 r NUTLIN 10µM, UKF-NB-3 r NUTLIN 10µMcontrol, or UKF-NB-3 r NUTLIN 10µMwtp53 cells, detecting p53 (Alexis Biochemicals via AXXORA Deutschland, Lörrach, Germany) and β-actin as loading control (Sigma, Munich, Germany).

5 MATERIAL AND METHODS Cloning of lentiviral vectors. Standard molecular cloning techniques were used to generate viral vectors based on Lentiviral Gene Ontology (LeGO) vectors [1, also Maps and sequence data of the vectors are available upon request. The human p53-cdna was kindly provided by Eva Lenfert, Department of Tumor Virology, Heinrich-Pette-Institute Hamburg. The 1182 bp cdna coding for human p53 gene (sequence identical to Accession No. NM_000546) was cloned by PCR into LeGO-iG2-Puro+ using EcoRI and NotI. The sequence of p53 was verified by sequencing. The internal SFFV promoter of this vector transcribes a bicistronic mrna with an internal ribosome entry site (IRES) of the Encephalo myocarditis virus (EMCV), expressing human wild type p53 together with a dual marker, the enhanced green fluorescent protein (egfp) coupled to a codon optimized puromycin resistance [2] by a P2A sequence. The monocistronic vector LeGO-G2-Puro+ expressing egfp and puromycin resistance coupled by a P2A sequence served as a control. Generation of viral particles. Cell-free viral supernatants were generated by transient transfection of 293T packaging cells as described [1], using the 3 rd generation packaging plasmids pmdlg/prre and prsv-rev [3] together with phcmv-vsv-g [4]. Supernatants containing pseudotyped vector particles were titrated on 293T target cells. Gene transfer rates were analysed 2 days after transduction by fluorescence activated cell scanning (FACS). Titres of 1 x10 7 (LeGO-iG2-Puro+-p53) and 2,5 x10 7 (LeGO-G2-Puro+) VSV-G pseudotyped vector particles per millilitre unconcentrated supernatants were obtained.

6 Cell culture and lentiviral gene transfer. All cells were cultured in their respective growth media supplemented with penicillin/streptomycin. For transduction of UKF-NB-3 r NUTLIN 10 target cells, 5x10 4 cells in 500µl medium were plated per well in a 24-well plate. The next day, 100µl and 250µl of viral supernatant per well were added and incubated for 3 days. Fluorescence Microscopy. Pictures were taken using an IX71 fluorescence microscope (Olympus, Hamburg, Germany). Fluorescence dye egfp was detected at 484/507nm (excitation/emission) Western Blot. Cells were lysed in Triton X sample buffer and separated by SDS-PAGE, as described before [5]. Proteins were detected using specific antibodies against β-actin (Sigma, Munich, Germany) or p53 (Alexis Biochemicals via AXXORA Deutschland, Lörrach, Germany), and were visualized by enhanced chemiluminescence using a commercially available kit (Amersham via GE Healthcare, Munich, Germany). Viability assay. Cell viability was tested by the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) dye reduction assay after 96 h incubation, modified as described before [6]. References [1] Weber K, Bartsch U, Stocking C, Fehse B. A Multicolor Panel of Novel Lentiviral Gene Ontology (LeGO) Vectors for Functional Gene Analysis. Mol Ther 2008;16:

7 [2] Weber K, Mock U, Petrowitz B, Bartsch U, Fehse B. Lentiviral gene ontology (LeGO) vectors equipped with novel drug-selectable fluorescent proteins: new building blocks for cell marking and multi-gene analysis. Gene Ther 2010;17(4): [3] Dull T, Zufferey R, Kelly M, et al. A third-generation lentivirus vector with a conditional packaging system. J Virol 1998;72: [4] Beyer WR, Westphal M, Ostertag W, von Laer D. Oncoretrovirus and lentivirus vectors pseudotyped with lymphocytic choriomeningitis virus glycoprotein: generation, concentration, and broad host range. J Virol 2002;76: [5] Michaelis M, Michaelis UR, Fleming I, Suhan T, Cinatl J, Blaheta RA, Hoffmann K, Kotchetkov R, Busse R, Nau H, Cinatl J Jr. Valproic acid inhibits angiogenesis in vitro and in vivo. Mol Pharmacol. 2004;65: [6] Michaelis M, Suhan T, Cinatl J, Driever PH, Cinatl J Jr. Valproic acid and interferonalpha synergistically inhibit neuroblastoma cell growth in vitro and in vivo. Int J Oncol 2004;25: