Generation of stable clones expressing or not expressing the mini-mucin Mini5B.

<p>(<b>A</b>) Schematic representation of the control Ires–Luc and Mini5B–Luc vectors. The insert encoding the Mini5B was made by a signal sequence, 11 TR of 29 aa, one R-end domain of 111 aa, and two CYS domains of 110 aa. (<b>B</b>) Using the luciferase activity assay, four stable clones expressing the Ires–Luc transgene (clones B2, C5, C7, and C10) and two stable clones expressing the Mini5B–Luc transgene (clones A3 and D6) were obtained. (<b>C</b>) <i>MUC5B</i> mRNA expression was studied by qRT–PCR (TaqMan) in MCF7 parental cell line (white box), in MCF7 Ires-Luc clone (grey box) and in Mini5B-Luc clone (black box). Expression levels were normalized to mRNA levels of 18S and shown as x-fold relative to the normalized expression of <i>MUC5B</i> gene in MCF7 parental cells. Relative amounts of target genes were calculated using the ΔΔCt method. Values are means ± standard deviation from four to six independent samples. (<b>D</b>) MUC5B immunofluorescence analysis of MCF7 parental cells, Ires–Luc and Mini5B–Luc clones. Mini5B was secreted at the cell surface of Mini5B–Luc clones. Nuclei were counterstained with propidium iodide. Scale bar 50 µm. (<b>E</b>) Expression of the Mini5B was evaluated by RT–PCR. A PCR product of 159 bp was detected only in the Mini5B–Luc clone. (<b>F</b>) Western blot analysis of lysates from Ires–Luc clones, Mini5B–Luc clones, and Mini5B–Luc CYS shRNA subpopulation. (<b>G</b>) Inhibition of Mini5B production detected by Western blotting was quantified. Mini5B production of the clone Mini5B–Luc was set at 100 and the production of Ires–Luc was set at 0.