Myoferlin in Breast Cancer EMT & Invasion Figure 4. Expression of EMT markers following myoferlin depletion in MDA-MB-231 cells. (A) Representative immunoblots (n = 3) of select EMT markers in lentiviral control (LTV-ctrl) and myoferlin depleted (MYOF-KD) cells serum-starved for 24 h. (B) Graphs illustrating the semi- quantitative evaluation of the expression of EMT markers by densitometry analysis of blots (n = 3). Density was presented in the graph as ‘‘relative density (%)’’ with density of lentiviral controls normalized to 100%, and statistical testing was done with the 1-sample t-test. doi:10.1371/journal.pone.0039766.g004 MMP1 mRNA could be down-regulated as an off-target effect of the RNAi targeting construct. We therefore screened additional MYOF-targeting shRNA constructs. Although two constructs (Figure S3) yielding partial MYOF knockdown (mean 64–66% knockdown compared with controls, n = 4) were not associated with reductions in MMP1 (data not shown), an additional construct (#320398) that targets the coding sequence of MYOF (Figure S3) resulted in a mean of 91% MYOF knockdown (n = 3) and was also associated with significant reductions in MMP1 release (Figure S7A and B). In addition, we performed an extensive in silico analysis to examine potential off-target effects of construct #10628. A search through the human nucleotide database for sequences similarity to the 21 nucleotide sequence of #10628 did not reveal MMP1 as a transcript with significant alignment (BLASTN version 2.2.24+) [42]. A search through the microRNA database TargetScan Custom (http://www.targetscan.org/, release 5.1, accessed Octo- ber 10, 2010) using the 2–8 mer sequence of ‘‘CTCTACT’’ from the #10628 construct did not show MMP1 to be a potential target. Construct #10628 is from a clone cataloged by the RNAi Consortium (http://www.broadinstitute.org), and a search through the consortium database did not show the MYOF shRNA construct as a non-targeting clone with matching transcripts in the MMP1 gene. Finally, the construct sequence was placed into the miRBase [43] to search against known miRNAs. There were no matches of the sequence against mature miRNAs, and all matches to stem-loop sequences using the SSEARCH method were from non-human species (silk worm, mosquito, and platypus) and were not significant (E-value ..0.01). We did additional PCR screening using a commercial extracellular matrix and adhesion molecules array to profile the expression of MMP genes (Table S2). The screen validated the MMP1 PCR results showing down-regulation of MMP1 mRNA in 231MYOF-KD cells. Specifically, of the four MMPs (MMP1, 10, 11, 14) expressed at reasonable levels in the MDA-MB-231 cells (i.e. PLoS ONE | www.plosone.org 6 June 2012 | Volume 7 | Issue 6 | e39766