Michael F. Reed, MD, William A. Zagorski, BS, John A

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Inhibition of Retinoblastoma Tumor Suppressor Activity by RNA Interference in Lung Cancer Lines Michael F. Reed, MD, William A. Zagorski, BS, John A. Howington, MD, Jack T. Zilfou, PhD, Erik S. Knudsen, PhD The Annals of Thoracic Surgery Volume 82, Issue 1, Pages 249-253 (July 2006) DOI: 10.1016/j.athoracsur.2006.02.033 Copyright © 2006 The Society of Thoracic Surgeons Terms and Conditions

Fig 1 Transfection of short-hairpin RNA targeted to retinoblastoma gene effectively diminishes expression of the retinoblastoma (RB) tumor suppressor (immunoblot analysis). β-Tubulin serves as a protein loading control. (MSCV = NCI H520 pMSCVpuro; Rb3C = NCI H520 pMSCVpuro-Rb3C). The Annals of Thoracic Surgery 2006 82, 249-253DOI: (10.1016/j.athoracsur.2006.02.033) Copyright © 2006 The Society of Thoracic Surgeons Terms and Conditions

Fig 2 Downregulation of retinoblastoma (RB) by means of small interfering RNA results in increased expression of retinoblastoma target genes (immunoblot analysis). (CDK2 = cyclin-dependent kinase 2; PCNA = proliferating-cell nuclear antigen; MSCV = NCI H520 pMSCVpuro; Rb3C = NCI H520 pMSCVpuro-Rb3C; TopoIIα = topoisomerase II alpha; TS = thymidylate synthase). The Annals of Thoracic Surgery 2006 82, 249-253DOI: (10.1016/j.athoracsur.2006.02.033) Copyright © 2006 The Society of Thoracic Surgeons Terms and Conditions

Fig 3 Inhibition of retinoblastoma activity by small inhibitory RNA increases in vitro growth rates in non–small cell lung cancer cells. NCI H520 cells harboring the retinoblastoma short-hairpin RNA (Rb3C), or the empty vector control (MSCV), were counted every 48 hours. The relative cell count is in comparison to the cell number at day 0. Error bars represent one standard deviation. The Annals of Thoracic Surgery 2006 82, 249-253DOI: (10.1016/j.athoracsur.2006.02.033) Copyright © 2006 The Society of Thoracic Surgeons Terms and Conditions

Fig 4 Non–small cell lung cancer xenografts with reduced retinoblastoma expression by small interfering RNA (Rb3C) exhibit increased growth in nude mice, compared with the empty vector control (MSCV). Tumor volumes were calculated once the tumor was measurable. Error bars represent one standard deviation. The Annals of Thoracic Surgery 2006 82, 249-253DOI: (10.1016/j.athoracsur.2006.02.033) Copyright © 2006 The Society of Thoracic Surgeons Terms and Conditions