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Supplementary Figures 1 - 3, Table 1 from SIRT1 Inactivation Evokes Antitumor Activities in NSCLC through the Tumor Suppressor p27

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posted on 2023-04-03, 16:29 authored by Lijia Zhu, Christine Y. Chiao, Katelyn G. Enzer, Alexander J. Stankiewicz, Douglas V. Faller, Yan Dai

Fig.S1: The level of p27Kip1 mRNA was unaffected by SIRT1 silencing. The mRNA levels of p27 were measured by quantitative RT-PCR analysis. 5 mg RNA extracted from SIRT1 silenced (shSIRT1) or shRNA control (shControl) H1299 and H460 cells. The mRNA levels of p27 are expressed relative to b-actin transcripts. Each experiment was performed in triplicate and repeated three times. The error bars represent the SEM. Fig.S2: SIRT1 silencing has no effect on apoptosis. Cell extracts were made from SIRT1- silenced and shRNA-control H1299 and H460 cells, and immunoblot analysis was performed with PARP and β-actin antibodies. Fig.S3: A. SIRT1 has no effect on p21 expression in both p53 wild type and p53 null cells. Cell extracts were made from SIRT1-silenced and shRNA-control H460 (p53+/+) and H1299 (p53-/-) cells, and immunoblot analysis was performed with anti-acetylated-p53, p21 and β-actin antibodies. B. p16 is deleted in NSCLC H460 and A549 cells. Cell extracts were made from SIRT1-silenced and shRNA-control H460 (p16-/-) and A549 (p16-/-) cells, or p16 wild type Hela cells. The immunoblot analysis was performed with anti-acetylated-p16, and β-actin antibodies. Table 1. The p27, p53 and p16 status in studied NSCLC cell lines.

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ARTICLE ABSTRACT

P27Kip1 (CDKN1B) regulates cellular proliferation and senescence, and p27Kip1 deficiency in cancer is strongly correlated with poor prognosis of multiple cancer types. Understanding the mechanism of p27Kip1 loss in cancer and the consequences of restoring p27Kip1 levels is therefore critical for effective management during therapy. Here, SIRT1, a class III histone deacetylase (HDAC), is identified as an important regulator of p27Kip1 expression. Mechanistically, SIRT1 reduces p27Kip1 expression by decreasing p27Kip1 protein stability through the ubiquitin–proteasome pathway. In addition, SIRT1 silencing suppresses non–small cell lung cancer (NSCLC) proliferation and induces senescence in a p27Kip1-dependent manner. Furthermore, SIRT1 silencing dramatically suppresses tumor formation and proliferation in two distinct NSCLC xenograft mouse models. Collectively, these data demonstrate that not only SIRT1 is an important regulator of p27Kip1 but also SIRT inhibition induces senescence and antigrowth potential in lung cancer in vivo.Implications: SIRT1 is a key regulator of p27 protein levels and SIRT1 inhibition is a viable strategy for NSCLC therapy by means of p27 reactivation. Mol Cancer Res; 13(1); 41–49. ©2014 AACR.

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