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. 2008 Jan 3;177(8):861–870. doi: 10.1164/rccm.200708-1269OC

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Figure 4. — Decreased levels of sirtuin (SIRT1) protein and mRNA expression by cigarette smoke extract (CSE) treatment in monocyte–macrophage (MonoMac6) cells. (A) Western blots of soluble nuclear proteins (30 μg) extracted from CSE-exposed MonoMac6 cells. Expression of SIRT1 was determined using mouse monoclonal anti-SIRT1 antibody. The purity of nuclear extract was shown by the presence of lamin B (nuclear envelope protein) and the absence of the cytoskeletal protein α-tubulin (bands not shown). Gel pictures shown are representative of at least three separate experiments. (B) Densitometric values of SIRT1 were normalized against the loading control, β-actin. The relative level (% of control) of SIRT1 in MonoMac6 cells showed decreased levels of SIRT1 protein in response to CSE at 4 and 24 hours. (C) CSE decreased the levels of SIRT1 mRNA in MonoMac6 cells. After 4 and 24 hours of CSE treatment, total RNA was extracted from MonoMac6 cells using RNeasy kit (Qiagen, Germantown, MD). Reverse transcriptase–polymerase chain reaction was performed. Amplified products (SIRT1: 200 bp; GAPDH: 600 bp) were resolved by 1.5% agarose gel electrophoresis, stained with ethidium bromide. SIRT1 mRNA expression was decreased after 24-hour exposure to low concentrations of CSE (0.5 and 1%) compared with control. Data represent mean ± SEM of three experiments (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, significant compared with control values.

Figure 4. — Decreased levels of sirtuin (SIRT1) protein and mRNA expression by cigarette smoke extract (CSE) treatment in monocyte–macrophage (MonoMac6) cells. (A) Western blots of soluble nuclear proteins (30 μg) extracted from CSE-exposed MonoMac6 cells. Expression of SIRT1 was determined using mouse monoclonal anti-SIRT1 antibody. The purity of nuclear extract was shown by the presence of lamin B (nuclear envelope protein) and the absence of the cytoskeletal protein α-tubulin (bands not shown). Gel pictures shown are representative of at least three separate experiments. (B) Densitometric values of SIRT1 were normalized against the loading control, β-actin. The relative level (% of control) of SIRT1 in MonoMac6 cells showed decreased levels of SIRT1 protein in response to CSE at 4 and 24 hours. (C) CSE decreased the levels of SIRT1 mRNA in MonoMac6 cells. After 4 and 24 hours of CSE treatment, total RNA was extracted from MonoMac6 cells using RNeasy kit (Qiagen, Germantown, MD). Reverse transcriptase–polymerase chain reaction was performed. Amplified products (SIRT1: 200 bp; GAPDH: 600 bp) were resolved by 1.5% agarose gel electrophoresis, stained with ethidium bromide. SIRT1 mRNA expression was decreased after 24-hour exposure to low concentrations of CSE (0.5 and 1%) compared with control. Data represent mean ± SEM of three experiments (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, significant compared with control values.