Myoferlin in Breast Cancer EMT & Invasion Figure 2. Morphology change following myoferlin depletion in MDA-MB-231 cells. Immunofluorescence micrographs showing the morphology of MDA-MB-231 wild type (WT), lentiviral transduction control (LTV-ctrl), and myoferlin knockdown (MYOF-KD) stable cell lines in culture. Note the more epithelial morphology of MYOF-KD cells compared to the more mesenchymal appearance of the WT and LTV-ctrl cells. Bar = 50 mm. doi:10.1371/journal.pone.0039766.g002 EMT markers, we found that the mRNA levels appeared to correlate with the Western results in that fibronection mRNA was down-regulated 3.46-fold while that of E-cadherin was up- regulated 2.58-fold in 231MYOF-KD cells compared with controls, although no significant change was found for vimentin (Table S1). Myoferlin Depletion Does Not Alter Cell Proliferation or Cell Cycle Distribution We next subjected 231LTV-ctrl and 231MYOF-KD cells to proliferation assays. First, to assess general proliferative status, MTS assays were conducted on wild-type, 231LTV-ctrl, and 231MYOF-KD cells. We observed no change in the growth rates of the three cell types (Figure S4A), indicating that MYOF does not profoundly alter tumor cell growth. To verify this result, we then carried out cell cycle analysis on the three cell types. Again, we detected no change in cell cycle activity in MYOF-depleted cells compared to wild-type or 231LTV-ctrl cells (Figure S4B). Myoferlin Depletion Attenuates Cell Invasiveness MYOF deficiency was not associated with a change in chemotaxis (Figure 5A) in Boyden chamber assays when 10% fetal bovine serum was used as the chemoattractant. However, 231MYOF-KD cells exhibited a significant ,38% reduction in invasiveness into Matrigel relative to 231LTV-ctrl cells (Figure 5B). These results suggest that targeted disruption of MYOF expression selectively attenuates invasiveness through reconstituted basement membrane matrix, with little effect on chemotactic migration. Myoferlin Depletion Alters MMP Expression The reduced invasive capacity of 231MYOF-KD cells led us to study whether proteins associated with tumor-microenvironment interactions may be altered following MYOF depletion. Specifi- cally, given that MYOF has been implicated in exocytosis [37], we hypothesized that its depletion might decrease the secretion of matrix metalloproteinases (MMPs) and/or tissue inhibitors of MMPs (TIMPs), as these are vital endopeptidases for degrading PLoS ONE | www.plosone.org 4 June 2012 | Volume 7 | Issue 6 | e39766