Expression of <i>TUSC1</i> mRNA and protein in cell lines.

<p>(A–B) The tumor cell lines (H290 and Nu6-1) were stably transfected with the <i>TUSC1</i> gene in pcDNA3.1 vector and CHO cells were transfected with <i>TUSC1</i> in pcDNA3.1/V5-His vector. <i>TUSC1</i> mRNA was amplified (RT-PCR) and exogenous protein (Western blot) was detected in all stably transfected cell lines containing <i>TUSC1</i> expression clones but not the parental cell lines transfected with empty vector. (C) Western blot analysis of endogenous TUSC1 proteins in tumor cell lines. Red box: Cell lines with homozygous deletion of <i>TUSC1</i>; Green box: cell lines with reduced TUSC1 expression but without <i>TUSC1</i> deletion. As an internal control for the amount of protein loaded, the same membrane was incubated with Anti-GAPDH antibody.</p

Representative immunohistochemical analysis using the TUSC1 antibody in normal and primary lung tissues.

<p>(A) Bronchioepithelium of Normal Lung. The basal layer was not positive for TUSC1 but differentiated cells in the upper layers of epithelium have high TUSC1 expression. Distribution of TUSC1 staining was cytoplasmic, nuclear and nuclear/cytoplasmic in the cells. (B, C) Lung cancer tissue microarray stained with <i>TUSC1</i> antibody and representative tissue cores (1–3) for each level of staining intensity. (D–F) Kaplan-Meier survival plots for the overall patient (D), adenocarcinoma (E) and squamous cell carcinoma (F). Survival status and the associated <i>P</i> value are indicated. Red line represents tumors with higher expression levels of <i>TUSC1</i> (score: 3), green line represents tumor with lower expression levels of <i>TUSC1</i> (score: 0–2).</p

Immunofluorescent staining by confocal microscopy.

<p>Localization of <i>TUSC1</i> in stable transfectants of CHO and 9HTE cell lines. (A) Stably transfected CHO cells were incubated with V5 or <i>TUSC1</i> antibodies. (B–C) Un-transfected 9HTE and SKEMS-1 lung cancer cells were incubated with <i>TUSC1</i> antibody. Subcellular distributions of <i>TUSC1</i> proteins are cytoplasmic and nuclear. V5 antibody was detected with Texas Red-conjugated anti-mouse IgG (Red) and <i>TUSC1</i> antibody was detected with either Texas Red-conjugated anti-rabbit IgG (Red) or by FITC-conjugated anti-rabbit IgG (Green).</p

Overexpression of <i>TUSC1</i> reduces cell growth and tumorigenicity of lung cancer cells.

<p>(A, B) <i>In vitro</i> cell growth of cancer cell lines (Nu6-1 and H290) transfected with <i>TUSC1</i>. (C, D) Tumor volume of subcutaneously injected stably transfected cancer cells with <i>TUSC1</i> in nude mice <i>in vivo</i>. Restoration of <i>TUSC1</i> expression in cell lines with <i>TUSC1</i> homozygous deletion suppressed cell growth and tumor development compared to the parental cell lines transfected with the empty vector (p<0.05 ).</p