Single-knockout phenotypes in Jeg-3 cells. (A) Analysis of protein expression of ERM proteins and LOK/SLK kinases in Jeg-3 single and double CRISPR cell lines. Analysis was based on Western blots using specific primary antibodies for LOK, SLK, radixin, and ezrin. Band intensity measurements were calculated in ImageJ and normalized to tubulin loading control. No significant differences between protein expression were found, suggesting that there is no upregulation of individual ERMs or LOK/SLK kinases in the absence of each. n = 3. (B) Growth curve wild-type (gray) versus Ezr^−/− Rdx^−/− (blue) and LOK^−/− SLK^−/− cells (orange). n = 15 wells of cells shown, wild type; n = 15, Ezr^−/− Rdx^−/−; n = 15, LOK^−/− SLK^−/−. 3,000 cells were seeded per well on 96-well plates and imaged every hour using the Incucyte live-cell analysis system. Each point represents cell confluence, measured by Incucyte over time per hour from 0 to 100 h. Error bars, SEM. (C) Representative immunofluorescence staining of microvilli in single-knockout cells used for microvilli quantification and assessment of partial defects. Scale bars, 10 µm. (D) Western blots showing expression of ezrin and LOK constructs in Jeg-3 cells. Asterisk indicates the presence of a nonspecific band.