CD133 expression in MDA-MB-231 cells. (A) CD133 surface expression evaluated in MDA-MB-231 cells by means of flow cytometry after staining with CD133/2 (293C3) and CD133/1 (AC133) phycoerythrin conjugated antibodies and with a hybridoma supernatant (clone 7). The expression of each antigen is represented on a frequency distribution histogram (count vs. PE signal). The open histograms, outlined by coloured lines, represent positive staining for CD133 and gray filled histogram shows negative control stained with matched isotype antibody. (B) The surface expression of CD133, measured with the indicated antibodies, is presented on a biparametric dot plot. The quadrants are gated to separate positive populations. The percentage of cells expressing high levels of CD133 is indicated at the upper right of each panel, together with their mean fluorescence intensity (MFI). (C) Western blot analysis with the anti-CD133 antibody on CD133 immunoprecipitated from MDA-MB-231 cells transfected with non-silencing RNAs (Ctrl siRNAs) or with siRNAs specific for CD133 (CD133 siRNAs). Lysates from the same cells were analyzed for β-tubulin content, as internal control of processed proteins. (D) Cytofluorimetrical analysis of CD133 expression performed in MDA-MB-231 cells transfected with non-silencing RNAs (Ctrl siRNAs) or with siRNAs specific for CD133 (CD133 siRNAs). The open histograms, outlined by coloured lines, represent positive staining for CD133 and gray filled histograms show negative controls stained with matched isotype antibody. (E) CD133 surface expression measured with PE-conjugated 293C3 antibody in MDA-MB-231 cells cultured in the presence of Tunicamycin for 24 hours and shown as dot plots in which the percentage of positive cells and their mean fluorescence intensity (MFI) are indicated at the upper right. The data are representative of three separate experiments.