Journal: Experimental Cell Research

Article Title: Up-regulation of 12(S)-lipoxygenase induces a migratory phenotype in colorectal cancer cells

doi: 10.1016/j.yexcr.2011.12.017

Figure Lengend Snippet: Differential expression of cell surface molecules. a: RNA was isolated from Caco2-LOX and Caco2-co cultures and expression of E-cadherin, integrin α5 and integrin β1 determined by qRT-PCR. # and ## represent a decrease as compared to control at p < 0.05 and 0.01 by Mann–Withney test. b, c: RNA was isolated for the determination of E-cadherin (b) and integrin β1 (c) expression by qRT-PCR. Results are shown relative to GAPDH expression. ### indicates a decrease as compared to Caco2 at p < 0.001 by unpaired t -test. d, e: Untransfected cultures (Caco2, SW480, SW620) and Caco2 transfectants (Caco-co, Caco-LOX) were harvested at semi-confluence. In Caco-LOX cells 12(S)-LOX expression was knocked down by lipofection with an anti-sense oligonucleotide (as-scr Caco-LOX and as-LOX Caco-LOX) and cultures were used at the time-point of minimal 12(S)-LOX expression. E-cadherin (d) and integrin β1 (e) on the cell surface was determined by FACS analysis. The mean fluorescence intensity was used as a measure of protein expression. The data were normalised to Caco2 or the respective control for better comparison. All data represent pooled results from 3 independent experiments using duplicate measurements. #, ##, ### indicate a decrease as compared to the respective controls at p < 0.05; 0.01 or 0.001. * represents an increase at p < 0.05 by Mann–Withney test. f: Cultures were grown to semi-confluence and then homogenised in lysis buffer for determination of integrin β1 by western blotting. The figure depicts representative blots above the column of semi-quantitative assessment of band intensity. Results are pooled from at least 2 experiments using duplicate lanes and # indicates a decrease at p < 0.05. g: SW620 and SW480-LOX cells were grown to semi-confluence and then exposed to 0.7 μM or 6 μM baicalein in serum-free medium. Protein was harvested and integrin β1 level determined as described above. All data points represent the mean ± SEM of 2 independent experiments using duplicate cultures. * increased above control at p < 0.05.

Article Snippet: Detection of integrin β1 was done using a PE-labelled antibody recognising total integrin β1 (clone MAR4, Becton Dickinson).

Techniques: Quantitative Proteomics, Isolation, Expressing, Quantitative RT-PCR, Control, Fluorescence, Comparison, Lysis, Western Blot