Statistical

Analysis All experiments were carried out with a buy CX-6258 minimum of n = 3. Intergroup comparisons were made by Student’s t test with P < 0.05 considered statistically significant. Results Expression of UCH-L1 in non-small cell lung carcinoma lines To identify a cell line model exhibiting high UCH-L1 expression that could be modulated for further investigations a range of human non-small cell lung carcinoma cell lines was surveyed for UCH-L1 expression by q-PCR and immunoblotting and compared to a normal lung cell line BEAS-2B (Figure 1). This revealed several EPZ015938 cell line cell lines (H157, H460 and H838) with high levels of UCH-L1 mRNA expression (Figure 1A). Interestingly, the cell lines with elevated UCH-L1 expression had differing origins; H460 is a large cell lung carcinoma while H157 is of squamous cell origin and H838 is an adenocarcinoma established from a metastatic lymph node. The level of UCH-L1 protein was found to reflect mRNA expression shown in Figure 1B & 1C, with H157, H460 and H838 exhibiting abundant protein production. Sequencing the UCH-L1 gene in these different cell lines failed to detect

any mutations. Cell blocks of H157 and H838 cells were also stained by immunohistochemistry for UCH-L1 expression and both stained positive for the protein (Figure 2A and 2B). Figure 1 UCH-L1 expression is higher in NSCLC cell lines than in normal lung cells. A. Fold change of UCH-L1 mRNA in lung cancer cell lines compared to the normal lung cell line BEAS-2B (n = 3). B. Densitometry of the level of UCH-L1 protein detected by Western Blot relative to the level of β-actin detected (n = 3). C. Nutlin-3a manufacturer Western Blot detection of UCH-L1 protein and β-actin loading control in different cell lines. Lanes as follows: 1 = H23, 2 = H157, 3 = H460, 4 = H838, 5 = BEAS-2B,

6 = MPP-89, 7 = REN, 8 = SKMES, 9 = UT-7. Figure 2 Immunohistochemistry showing UCH-L1 positive cells in H157 and H838 cells. Brown staining Ergoloid indicates the presence of UCH-L1 in H157 (A) and H838 (B) cells. (Scale bar is equivalent to 15 μm). Silencing of UCH-L1 expression in the H838 and H157 cell lines To establish if elevated UCH-L1 levels contribute to lung carcinogenesis, expression in H157 and H838 cells was silenced using siRNA and any subsequent phenotypic changes were investigated. UCH-L1 mRNA was substantially down-regulated in H838 cells at 24 hr post-transfection and remained decreased at 96 hr post-transfection (Figure 3A). Immunoblotting confirmed UCH-L1 protein was significantly reduced at 24 hr post-transfection and by 72 hr the protein was undetectable in both H838 cells (Figure 3B) and H157 cells (Figure 3C). Figure 3 Knockdown of UCH-L1 in H838 and H157 cells by siRNA. A. Percentage knockdown of UCH-L1 mRNA in H838 cells at 24 hr, 48 hr, 72 hr and 96 hr post-transfection compared to time-matched control. B & C. Immunoblot detection of UCH-L1 protein expression at 24 hr, 48 hr and 72 hr post-transfection in H838 cells (B) and H157 cells (C).