Inhibition of cell proliferation by ANO1 knockdown.

<p>(A) RNAi knockdown of ANO1 protein expression in ANO1 expressing cells was shown by Western blot images. The membrane proteins extracted from GLC82 and NCI-H520 cells on the 3rd day after transfection of different ANO1 shRNAs (shRNA1, shRNA2 and shRNA3) were immunoblotted with ANO1 antibody. ANO1 shRNA1 showed the most effective inhibition of ANO1 expression, compared with the other two shRNAs and the scrambled shRNA. (B) GLC82 or NCI-H520 cell proliferation was assessed by CCK8 assay based on the extracellular decreasing of WST8 by NADH produced in mitochondria. Using a microplate reader, the number of cells was quantified by the measurement of absorbance at 450 nm. Transfection of ANO1 shRNA1 resulted in significant inhibition of GLC82 and NCI-H529 cell viability in time-dependent manner as compared with cells treated with scrambled shRNA (n = 6). (C) Cell proliferation of carcinoma cells was assessed by the colony formation assay in culture in the presence of ANO1 shRNA1. Quantitative analysis of ANO1 silencing group showed less colony genesis, compared with scrambled shRNA group (n = 3). Representative images are showed below bar charts. (D) RNAi of ANO1 inhibits the clonogenicity of GLC82 cells in soft agar (n = 3). NCI-H520 failed to form colonies in soft agar. Statistical significance by Student’s <i>t</i>-test is indicated as *<i>p</i>< 0.05; **<i>p</i>< 0.01 and ***<i>p</i>< 0.001. Data are expressed as mean ± s.e.m.