Timm S, Titus B, Bernd K, Barroso M. general conclusions about the intrusive properties of the bulk of the cells in the assay. Recent advances in 3-dimensional (3D) culture techniques allow for the growth of tumor cells embedded in Matrigel or other ECM components. Using this method, whole colonies of tumor cells can be visualized, allowing researcher to make stronger conclusions about the behavior of the bulk of cells within a populace. Thus, we assayed tumor invasion and protease secretion using a Dye-quenched (DQ)-collagen IV 3D culture assay. DQ collagen IV fluoresces upon proteolytic cleavage and is a measure for the activation of many proteases, including lysosomal cathepsins [33,34]. Epithelial, NT mesenchymal, NT mesenchymal cells in the presence of EIPA, or Arl8b KD mesenchymal DU145 cells were Atractyloside Dipotassium Salt seeded in Matrigel supplemented with DQ-collagen IV and produced for 4 days in complete media. Cells were fixed and stained for actin (red) (Fig. 3C). Control-treated epithelial DU145 cells TSPAN33 formed spheroid colonies and had minimal protease activity, while control-treated mesenchymal DU145 cells formed highly invasive colonies Atractyloside Dipotassium Salt and displayed strong protease activity indicated by ample DQ-collagen IV fluorescence (green). In contrast, mesenchymal cells treated with EIPA or Arl8b shRNA formed spheroid colonies with minimal protease activity similar to the epithelial cell control. One explanation for the smaller colony size of Arl8b KD cells compared to NT cells is usually that Arl8b knockdown results in slower cell proliferation; however, loss of Arl8b had no effect on the proliferation rate of mesenchymal cells (Fig. S3). In addition to having higher protease activity in 3D culture, immunoblot revealed that mesenchymal DU145 cells also had increased expression of lysosomal cathepsins B, D, and L compared to epithelial DU145 cells (Fig. S4), which may partially account for the increased matrix degradation observed in mesenchymal-control treated colonies. Collectively, these data indicated that peripheral lysosome positioning regulates EMT-mediated tumor cell invasion and protease secretion as assayed in both a Boyden chamber and 3D culture system. Transforming growth factor beta (TGF) is usually another common component of the tumor microenvironment known to induce EMT and invasive potential of cancer cells [40]. We queried whether TGF would stimulate anterograde lysosome trafficking and invasion of the parental DU145 cells. Upon treatment with TGF, parental DU145 prostate cancer cells lost cell-to-cell adhesions and lysosomes were redistributed from the perinuclear region to being diffuse throughout the cytoplasm (Fig. S5A). Furthermore, TGF treatment resulted in increased invasion of parental DU145 cells in a Boyden chamber assay (Fig. S5B). Together, these data suggest that anterograde lysosome trafficking correlates with invasive potential in an inducible model of EMT. NHE1 is necessary for EMT-mediated anterograde lysosome trafficking NHE1 is usually often upregulated in invasive cancers and its activity is usually associated with anterograde lysosome trafficking and tumor cell invasion [17,21]. Immunoblot revealed that NHE1 protein levels Atractyloside Dipotassium Salt were increased in DU145 mesenchymal cells compared to epithelial cells (Fig. 4A; quantified in 4B). We previously showed that treatment with EIPA, a broad NHE inhibitor, resulted in juxtanuclear lysosome aggregation (Fig. 2B). To test whether NHE1 was important in regulating EMT-mediated lysosome trafficking, we used lentivirus- delivered shRNA to generate NHE1 KD mesenchymal DU145 cells. Immunoblot revealed that NHE1 levels were depleted by greater than 90%, and that NHE1 KD cells maintained a loss of E-cadherin, suggesting that NHE1 KD does not reverse EMT (Fig. 4C). Next, lysosomes (red) were visualized by immunofluorescence microscopy and this revealed that shRNA-depletion of NHE1 resulted in JLA (Fig. 4D; quantified in 4E). These data suggested that NHE1 regulates EMT-mediated anterograde lysosome.