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BioMed Research International
Volume 2014, Article ID 783459, 12 pages
Research Article

SIRT1 Inhibition Affects Angiogenic Properties of Human MSCs

1Department of Biochemistry, Biophysics and General Pathology, Second University of Napoli, Via L. De Crecchio 7, 80138 Napoli, Italy
2Istituto Nazionale Tumori, Struttura Complessa Oncologia Medica Melanoma Immunoterapia Oncologica e Terapia Innovativa, Via Mariano Semmola, 80131 Napoli, Italy
3Institute of Genetics and Biophysics “A. Buzzati-Traverso”, CNR, Via P. Castellino 111, 80131 Napoli, Italy

Received 18 July 2014; Revised 7 August 2014; Accepted 8 August 2014; Published 27 August 2014

Academic Editor: Zongjin Li

Copyright © 2014 Botti Chiara et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Supplementary Material

Supplementary Figure 1

Primary hMSCs were infected with HPV16 E6-E7 and hTERT lentiviral vectors expressing pSin hTERT and pSin E6-E7 [30] using a multi-infection program, as reported in Methods section. Two clones were obtained and tested for the presence of hTERT and E6-E7 transcripts. Based on RT-PCR data, both clones (MeBM1E1, MeBM1E2) showed similar levels of hTERT and E6-E7 transcripts. No hTERT and E6-E7 expression were detected in untransduced hMSCs (Supplementary Figure 1(a)). The resulting cell lines maintained a fibroblast-like phenotype comparable to primary hMSCs and showed no differences in hMSCs markers expression, such as CD73, CD90, and CD105 (Supplementary Figure 1(b)).Thus, these immortalizedmesenchymal cells (MeBM1E1, MeBM1E2) represent a valuable model that can be used for basic studies of mesenchymal biology.

Supplementary Figure 1 (Legend): A) RT-PCR analysis was performed on MeBM1E1 and MeBM1E2 clones to detect expression of hTERT and E6-E7 transcripts and (1B) different hMSCs markers expression compared to untransducted cells. M= molecular weight marker

Supplementary Figure 2

Genetic inhibition was obtained by silencing SIRT1 with lentiviral vector expressing sh-Sirt1-GFP. In order to determine the infection efficiency, green fluorescent protein (GFP) was monitored using fluorescence microscopy after 10 days from infection. As shown in Supplementary Figure 2(a), high levels of GFP in sh-Sirt1- hMSCs were observed with a concomitant reduction of SIRT1 protein (Supplementary Figure 2(b)).

Supplementary Figure 2 (Legend): A) Representative picture of high levels of GFP in sh-Sirt1-hMSCs (40x magnification, Scale bars=50 µm). B) SIRT1 and tubulin were analyzed by Western blotting in hMSCs and sh- Sirt1-hMSCs.

  1. Supplementary Figures