Myoferlin in Breast Cancer EMT & Invasion Figure 3. Morphology change following myoferlin depletion in MDA-MB-231 cells. Atomic force and scanning electron microscopy images showing the spindle, elongated shape of lentiviral control (LTV-ctrl) cells and the more flat and circular morphology of myoferlin depleted (MYOF-KD) cells. AFM imaging shows pronounced actin stress fibers (black arrows) oriented along the long axis being evident in the control but not in the MYOF depleted cells. Cytoplasmic poles, lamellipodia and filopodia are observable in the SEM images. White arrowheads indicate the leading edge of cells. doi:10.1371/journal.pone.0039766.g003 ECM constituents [38]. To that end, we collected 24 hour conditioned, serum-free supernatants from 231LTV-ctrl and 231MYOF-KD cells and performed an array-based multiplex sandwich ELISA to screen the expression of several of these endopeptidases and their regulators (Figure S5). We observed a reduction in the secretion of MMP-1, -2, -3, and -8 from 231MYOF- KD cells (MMP-2, -3, and -8 were undetectable in 231MYOF-KD cells). MMP-13 levels were not drastically affected, while in contrast, levels of MMP-9 and -10 were elevated in 231MYOF-KD cells. In addition, the 231MYOF-KD cells had reduced levels of TIMP-4, but no notable changes in TIMP-1 and -2 releases were evident. Of the detectable MMPs, the most pronounced change was the decrease in secreted MMP1. However, given the small sample size of this screening experiment (n = 1), statistical analysis could not be performed to validate the significance of the observed fold changes. Therefore, to validate the change in MMP1, multiple biological replicates of concentrated 24 hour supernatant from 231LTV-ctrl and 231MYOF-KD cells were subjected to immunoblotting (Figure 6A), and secreted MMP1 was verified to be decreased in 231MYOF-KD cells compared to controls. We also confirmed the diminution (,97%) of pro-MMP1 secretion into the extracellular milieu by solution-phase ELISA of 231LTV-ctrl and 231MYOF-KD cells (Figure 6B). To confirm the functional association between MYOF knockdown and MMP1 depletion, we investigated the invasive capacity of 231MYOF-KD cells through a collagen I matrix, since fibrillar collagen is a key substrate for the MMP1 enzyme [39] and Matrigel lacks collagen I [40]. In support of these previous observations, 231MYOF-KD cells exhibited a ,62% reduction in invasion through collagen I matrix compared with 231LTV-ctrl cells (Figure 6C). We next tested whether MMP1 expression was diminished at the transcriptional level using qRT-PCR. Surprisingly, MMP1 mRNA levels were reduced over an average 18.8-fold in 231MYOF- KD cells when compared with the 231LTV-ctrl cells (Figure S6). This finding of MMP1 mRNA down-regulation in 231MYOF-KD cells was striking, as there have been no reports from other laboratories suggesting that MYOF may contribute to gene expression regulation. Since the diminution of MYOF in the MDA-MB- 231 cells was mediated by shRNA (which mimics the function of microRNA molecules [41]), we recognized the potential that PLoS ONE | www.plosone.org 5 June 2012 | Volume 7 | Issue 6 | e39766