Isoform-specific knockdown and absence of significant proliferation, viability and morphological effects.

<p>(<b>A</b>) MCF10A cells were transfected with siRNA oligonucleotides (20 nM) targeting individual isoforms of <i>LAMA3</i> (left panel) or <i>PTHLH</i> (right panel), siRNA pools targeting all isoforms (‘gene-level’) or with control scrambled siRNA oligonucleotides (sictrl denotes siRNA control, ‘i1,o1’ denotes the first oligonucleotide targeting isoform 1; ‘i2,o2’ the second oligonucleotide targeting isoform 2, etc. and ‘GL’ denotes ‘gene level’). Forty-eight hours later, RNA was extracted and qPCR measurements were performed using primers specific to all expressed individual isoforms, as well as primers amplifying transcript regions common to all expressed isoforms. Measurements were normalized to results obtained with an independent siRNA control replicate. Error bars denote standard errors from three technical replicates and asterisks denote statistically significant (p<0.05) differences relative to siCtrl. Similar results were obtained in three independent repeat experiments. (<b>B</b>) Oligonucleotides for <i>EGFR</i>, which impair migration, and for <i>CSNK1G2</i>, which accelerate migration are shown as additional controls. Cell cycle analysis was performed as described in <i>Methods</i>. Error bars represent the standard errors from the analysis of 24 images taken per from each of four wells per condition. (<b>C</b>) Viability was measured by tetrazolium-based WST-1 assay. MCF10A cells were transfected with the indicated oligonucleotides (siRNA to polo-like kinase, <i>PLK1</i>, served as positive control). Error bars represent standard errors from 3 biological replicates. The experiment was performed thrice. (<b>D</b>) Shown are morphologic effects of isoform-specific oligonucleotides targeting <i>LAMA3</i> or <i>PTHLH</i>. Nine thousand MCF10A cells per well were transfected, starved and stimulated as described for the proliferation assays, followed by DAPI and phalloidin staining, 14 hours after stimulation. Automated image analysis was used to systematically assess changes in cell size and shape by analyzing 24 images taken per condition. No difference between the individual knockdowns was apparent (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080566#pone.0080566.s006" target="_blank">Figure S6B</a>); one representative image for each condition is shown.