Knockdown of EF1 causes variable changes in morphologies as a function of tissue microenvironment. (a) Western blot shows efficient knockdown of EWS-FLI1 in cell culture conditions upon 72 h addition of doxycycline (DOX). (b) Images of TC32 cells expressing C shRNA or EF1 shRNA with or without addition of DOX in cell culture. A subset of cells express F-Tractin mRuby2 (red on left, gray on right), while all cells express the doxycycline-inducible cytoplasmic GFP (green), allowing for mosaic cell labeling. Mild changes in actin organization are observed with expression of EF1 shRNA. However, no clear distinctions in cell morphology are seen. (c) Similarity matrix based on pairwise permutation test of Tukey depth median of first two PCs shows distinct differences in cell states between all three tissue microenvironments in TC32 C shRNA (left). Upon loss of EF1, cell states in all three regions change significantly (right), and enviromentally dependent differences become less distinct (middle), suggesting expression of EF1 enables tissue-specific specialization of cells. (d and e) Loss of 50% of data (d), but not simulations of reduced axial resolution by Gaussian blurring in the z-dimension (σ = 3 pixels; e), leads to loss of distinction between experimental groups, as visualized by similarity matrix based on pairwise permutation tests. Source data are available for this figure: SourceData FS5.