Figure 6.
Knockdown of EF1 causes variable changes in morphologies as a function of tissue microenvironment. (a–c) Morphological cell states were examined as a function of tissue microenvironment (HBV, PVS, or CHT) in EwS cells expressing a dox-inducible scrambled (C, left, n = 18, 25, 28 for HBV/PVS/CHT, respectively) or EWSR1-FLI1 (EF1, right, n = 25, 26, 19 for HBV/PVS/CHT, respectively) targeting shRNA. Representative cell renderings from each group are color-coded by k-means clustering in Fig. 2 e. (d) Bagplots below display PCA of each cell line as a function of region (HBV, red, PVS, black, CHT, white). The inner polygon is constructed on the basis of Tukey depth, and contains at most 50% of the data points. The outer circle encompasses all data excluding outliers, which are marked by black asterisks. White plus sign represents the Tukey depth median. In C shRNA expressing cells, all three regions showed distinct cell states in PC space (left). Upon knockdown of EF1, cells are shifted to a more protrusive state and differences as a function of region are relatively reduced (right). (e) A subset of EwS C (e’) or EF1 shRNA (e’’) expressing cells express F-Tractin mRuby2 (white in top panel, red in bottom panel) in addition to dox-inducible cytoplasmic GFP (green, bottom panel), allowing for labeling of individual cells. EwS cells in HBV can maintain a pseudorosette-like organization upon EF1 knockdown, despite altered cell morphologies. (see also Video 2). (f) Proportion of contact with other TC32 cells expressing C shRNA or EF1 shRNA is plotted against sphericity. Upon EF1 knockdown, cells with distinctly low sphericity (<0.8, dashed line) could now also be observed with only partial contact to other cells. Scale bar, 20 µm.

Knockdown of EF1 causes variable changes in morphologies as a function of tissue microenvironment. (a–c) Morphological cell states were examined as a function of tissue microenvironment (HBV, PVS, or CHT) in EwS cells expressing a dox-inducible scrambled (C, left, n = 18, 25, 28 for HBV/PVS/CHT, respectively) or EWSR1-FLI1 (EF1, right, n = 25, 26, 19 for HBV/PVS/CHT, respectively) targeting shRNA. Representative cell renderings from each group are color-coded by k-means clustering in Fig. 2 e. (d) Bagplots below display PCA of each cell line as a function of region (HBV, red, PVS, black, CHT, white). The inner polygon is constructed on the basis of Tukey depth, and contains at most 50% of the data points. The outer circle encompasses all data excluding outliers, which are marked by black asterisks. White plus sign represents the Tukey depth median. In C shRNA expressing cells, all three regions showed distinct cell states in PC space (left). Upon knockdown of EF1, cells are shifted to a more protrusive state and differences as a function of region are relatively reduced (right). (e) A subset of EwS C (e’) or EF1 shRNA (e’’) expressing cells express F-Tractin mRuby2 (white in top panel, red in bottom panel) in addition to dox-inducible cytoplasmic GFP (green, bottom panel), allowing for labeling of individual cells. EwS cells in HBV can maintain a pseudorosette-like organization upon EF1 knockdown, despite altered cell morphologies. (see also Video 2). (f) Proportion of contact with other TC32 cells expressing C shRNA or EF1 shRNA is plotted against sphericity. Upon EF1 knockdown, cells with distinctly low sphericity (<0.8, dashed line) could now also be observed with only partial contact to other cells. Scale bar, 20 µm.

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