LoxP. SFFV mcherry wpre SIN-LTR. shrna-p53. LoxP. shrna-scr

Transcription

1 LeGO-C-p53-shRNA SIN-LTR? RRE cppt H1/TO shrna-p53 SFFV mcherry wpre SIN-LTR LeGO-C-scr-shRNA SIN-LTR? RRE cppt H1/TO shrna-scr SFFV mcherry wpre SIN-LTR Supplementary Figure 3A: Scheme of the lentiviral vectors, as integrated provirus, 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; H1/TO, RNApolymerase III promoter; shrna-p53, short hairpin RNA against human p53; shrna-scr, short hairpin RNA with no target (scrambled); SFFV, Spleen focus-forming virus enhancer/promoter; mcherry, a red fluorescent protein; wpre, Woodchuck hepatitis virus post-transcriptional regulatory element.

2 UKF-NB-3 r NUTLIN 10µM UKF-NB-3 r NUTLIN 10µM UKF-NB-3 r NUTLIN 10µMscr-shRNA UKF-NB-3 r NUTLIN 10µMscr-shRNA UKF-NB-3 r NUTLIN 10µMp53-shRNA UKF-NB-3 r NUTLIN 10µMp53-shRNA Supplementary Figure 3B: UKF-NB-3 r NUTLIN 10µM cells, UKF-NB-3 r NUTLIN 10µM cells transduced with LeGO-C-scr-shRNA (UKF-NB-3 r NUTLIN 10µMscr-shRNA ), and UKF-NB- 3 r NUTLIN 10µM transduced with LeGO-C-p53-shRNA (UKF-NB-3 r NUTLIN 10µMp53-shRNA ) cells

3 were photographed by inverse light microscopy (left column) and by fluorescence microscopy (right column), using an IX71 fluorescence microscope (Olympus, Hamburg, Germany). The fluorescence dye mcherry was detected at 587/610 nm (excitation/emission). No fluorescence was detected in the non-transfected control cell line UKF-NB-3 r NUTLIN 10µM. UKF-NB-3 r NUTLIN 10µMscr-shRNA and UKF-NB-3 r NUTLIN 10µMp53-shRNA cells displayed high fluorescence indicating succesful lentiviral transduction.

4 UKF-NB-3 r NUTLIN 10µM UKF-NB-3 r NUTLIN 10µMscr-shRNA UKF-NB-3 r NUTLIN 10µMp53-shRNA Mean: 44 Mean: 1082 Mean: ,6 % 77,7 % 76,8 % Supplementary Figure 3C: Flow cytometric analysis (BD FACS Canto II) of mcherry fluorescence (detected at 587 nm (excitation)/610 nm (emission)) in UKF-NB- 3 r NUTLIN 10µM, UKF-NB-3 r NUTLIN 10µMscr-shRNA, or UKF-NB-3 r NUTLIN 10µMp53-shRNA cells. The upper row shows the fraction of cells gated for fluorescence from forward scatter (FSC) vs. side scatter (SSC) dot plots. The middle row displays histograms showing cellular fluorescence and mean values of cellular fluorescence. The lower row expresses the amount of fluorescent cells (in %).

5 p53 UKF-NB-3 r Nutlin 10µM UKF-NB-3 r Nutlin 10µMscr-shRNA UKF-NB-3 r Nutlin 10µMp53-shRNA β-actin Supplementary Figure 3D: P53 expression in UKF-NB-3 r NUTLIN 10µM cells, UKF-NB- 3 r NUTLIN 10µMscr-shRNA cells, or UKF-NB-3 r NUTLIN 10µMp53-shRNA cells as indicated by Western blot using antibodies directed against p53 (Alexis Biochemicals via AXXORA Deutschland, Lörrach, Germany) and β-actin (Sigma, Munich, Germany) serving as loading control. P53 expression was strongly reduced in UKF-NB-3 r NUTLIN 10µMp53-shRNA cells relative to UKF- NB-3 r NUTLIN 10µM cells and UKF-NB-3 r NUTLIN 10µMscr-shRNA cells.

6 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 60 bp shrna against human p53 or the 60 bp scrambled shrna was cloned into LeGO-C using XbaI and NotI. The internal promoter H1/TO transcribes the shrna against p53 or the scrambled shrna, respectively. The internal SFFV promoter transcribes the red fluorescent protein mcherry as a marker. The vector LeGO-C only expressing mcherry served as a control. Generation of viral particles. Cell-free viral supernatants were generated by transient transfection of 293T packaging cells as described [2], using the 3 rd generation packaging plasmids pmdlg/prre and prsv-rev [3] together with phcmv-vsv-g [2]. Supernatants containing pseudotyped vector particles were titrated on 293T target cells. Gene transfer rates were analyzed 2 days after transduction by fluorescence activated cell sorting (FACS). Titers of 1,5 x10 7 (LeGO-C-p53-shRNA) and 2 x10 7 (LeGO-C-scr-shRNA) VSV-G pseudotyped virus particles per milliliter un-concentrated supernatants were obtained. 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 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 in the presence of 8µg/ml polybrene and the plate was centrifuged at 1000 g for 1 hour at room temperature. After another 3 hours in the cell culture incubator, medium was replaced.

7 Fluorescence Microscopy. Pictures were taken using an IX71 fluorescence microscope (Olympus, Hamburg, Germany). Fluorescence dye mcherry was detected at 587nm emission/610 nm excitation. Flow Cytometry. Cells were analysed on a FACSCanto II (BD, Heidelberg, Germany), using DIVA-software (BD, Heidelberg, Germany). Western Blot. Cells were lysed in Triton X sample buffer and separated by SDS-PAGE, as described before [4]. Proteins were detected using specific antibodies against β-actin (Sigma, Munich, Germany) or p53 (Alexis Biochemicals via AXXORA Deutschland, Lörrach, Germany), and were visualised by enhanced chemiluminescence using a commercially available kit (Amersham via GE Healthcare, Munich, Germany). 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: 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: Dull T, Zufferey R, Kelly M, et al. A third-generation lentivirus vector with a conditional packaging system. J Virol 1998;72: 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(3):520-7.