Figure 3.
The TAZ–TEAD interaction mediates MES transition in GSCs. (A) Western analysis of GSC11 clones overexpressing vector, 4SA, or 4SA-S51A showing Flag expression. Retrovirus encoding pBabe vector or TAZ mutants was used for transduction of GSC11. Stable clones were generated by culturing transduced cells in puromycin. (B) Immunoprecipitation of TAZ using Flag antibody followed by Western analysis of TAZ, TEAD4, and RUNX2 in 4SA- and 4SA-S51A-expressing cells. (C) Western analyses of MES proteins in GSC11 clones expressing vector, 4SA, and 4SA-S51A. (D) Invasion assay of GSC11 clones across Matrigel-coated transwell plates. Bar graphs indicate percentage of invasion. A t-test was used to compare statistical differences. (E) GSC11 clones were subjected to EdU incorporation assay; cells were plated on laminin/poly-L-ornithine-coated chamber slides, fixed, and counterstained with DAPI; and Alexa 488-positive cells were quantified using a fluorescent microscope in 10 independent fields. Bar graphs indicate mean values. A t-test was used to assess statistical significance. (NS) Not significant. (F) Western analyses of cell cycle proteins in GSC11 clones. (G) Western analyses of MES proteins after stable knockdown of TEADs. Two independent stable knockdown pools (shTEAD-a and shTEAD-b) were generated using pGipZ lentivirus. Both clones appeared to silence TEAD2 and TEAD4 effectively. (H) Osteoblast induction of GSC11 clones. Cells were cultured in differentiation medium for 3–4 wk, fixed in 70% ethanol, stained with Alizarin Red for assessing intercellular calcium deposition, and photographed. (I) Chondrocyte induction of vector control and 4SA. Cells were grown as pellets in chondrocyte medium for 4 wk, fixed in buffered 10% formalin, and embedded in paraffin. Five-micron were slide-mounted and stained for glycosaminoglycans with Safranin O.