As a subpopulation of malignancy cells exhibiting stem-like properties are considered to be critical for metastasis [58], detailing the role of K19 in malignancy stem cells will be important to study in the future. In the absence of K19, E-cadherin was found in endocytic compartments indicating that most E-cadherin was internalized in the absence of K19. cells upon detachment from your extracellular matrix remains unclear. In the context of breast malignancy, previous studies have shown that depletion of K19 increases cell migration and invasion knockout (KO) cells with re-expression of K19, we observed that K19 is required for the epithelial-like cell shape and proper cell-cell adhesion. These events were accompanied by lower levels of plakoglobin but accumulation of E-cadherin in endocytic compartments in the absence of K19. Importantly, while we confirmed the inhibitory role of K19 on cell migration and invasion, K19 was found to be required for cells to grow in low attachment conditions. Results KRT19 KO cells display an elongated phenotype Under the microscope, MCF7 KO cells showed a considerable difference in morphology from their parental counterpart. While parental (P) MCF7 cells were mostly epithelial-like and rounded in shape, KO cells exhibited more mesenchymal-like morphology with elongated and spindled designs (Physique 1aCFigure 1b). Of notice, two KO clones were used to confirm phenotypes associated with a loss of K19. To quantify the difference in designs between parental and KO cells, cells were sorted into two groups, rounded and elongated, based on their designs (Physique 1c). An elongated spindled cell shape with protrusions at cell edges was categorized as elongated while OTX008 a rounded morphology characterized by a circular cell shape was categorized as rounded. Scoring of cell designs confirmed that KO Rabbit Polyclonal to CDK5RAP2 cells were more elongated than parental cells as the majority of KO cells (54.4C72.3%) were elongated while the majority of parental cells (58.4%) were rounded in shape (Physique 1d). KO cells also exhibited decreased minor/major axis ratio (0.39C0.44) compared to parental cells (0.50), further verifying more elongated shape (Physique 1e). Circularities of KO cells were also less than that of parental cells (Physique S1A) and when a cutoff value of 0.48 was utilized for circularity (Figure S1B), the result mirrored what was observed in Figure 1d. Of notice, while two KO clones exhibited delicate differences from each other, both were more elongated than parental cells. Finally, digital holographic microscopy (DHM) was used to quantitate morphologies of parental and KO cells (Physique 1f). DHM measured cells based on index of refraction and physical thickness [33,34] and produced 17 parameters per single cell based on individual cell pseudoheight (models in nm) derived from phase maps (Table S1). Optical measurements included pixel mean, standard deviation, and texture parameters, while geometric parameters included roundedness of cells, eccentricity and circularity. A higher eccentricity and a lower circularity frequency confirmed the elongated phenotype of KO cells (Physique 1gCFigure 1h). Physique 1. Keratin 19 knockout cells display an elongated phenotype. (a) Whole cell lysates of parental (p) control and two different clones (KO1 and KO2) of KO cell lines were harvested, and immunoblotting OTX008 was performed with antibodies against the indicated proteins. Molecular weights in kDa. (b) Immunostaining of K18 (green) in P and KO cells. Nuclei are shown in blue. Bar, 20?m. (c) Phase-contrast images of representative rounded and elongated designs. Bar, 20?m. (d) Percentages of P and KO cells with rounded or elongated cell shape. Data from three experimental repeats are shown as mean??SEM. Students t-test: *p? ?0.05; **p? ?0.001. Chi-square test: p ?0.001. (e) Minor/Major axis ratios of P and KO cells. Data from four experimental replicates are shown as mean??SEM. Students t-test: *p? ?0.05; **p? ?0.001. (f) Phase pseudoheight maps of P and KO (KO2) cells collected by OTX008 digital holographic microscopy (DHM). Sum of squared deviation (SSD) shows the distance of individual cells from the population mean. Representative P and KO cells with the smallest SSD of 17 phase parameters collected shown. A color bar indicates cell phase height. Bar, 10?m. Histograms of (g) circularity and (h) eccentricity of P and KO cells from DHM analyses Weakened cell-cell adhesions in KRT19 KO cells In addition to elongated shape, KO cells were forming loose contacts between neighboring cells, whereas parental MCF7 cells were in close contacts with their neighbors (Physique 1b). To quantitate the difference, cell-cell adhesions made by subconfluent parental and KO cells were examined. Cell-cell adhesions were categorized into three different degrees: high indicates a cell attached to its neighboring.