Fig. 5. Etv2 knockdown abrogates the ability of endo-transdifferentiation of GBM tumor cells.
a Etv2 was knocked down using the CRIPR/Cas9 system in GBM-U87 cells. The sgRNA coding sequence is indicated in black, and the protospacer-adjacent motif (PAM) sequence is labeled in red. b Two ETV2 knockdown clones (E6 and E5) were selected, and the ETV2-mutation was confirmed by the T7 endo I assay. c The growth rate of the GBM tumor cells in vitro was not affected after ETV2 knockdown. d The tumor size of GBM-U87-E5 was significantly smaller than that of control GBM-U87 at 2 months after cell implantation (n = 5 in each group). e The expression level of ETV2 in subcutaneous GBM tumors was analyzed by WB. f ETV2 knockdown inhibited the induction of TDECs in GBM tumor cells in vivo. GBM-U87-E5 or GBM-U87 cells were subcutaneously implanted in NOD/Scid mice for 2 months (2 × 106 cells for each mouse, n = 5 per group). Multicolor IF staining for ki67, hCD31, and CD31 (recognizing both mouse and human antigen) revealed ki67+/hCD31+/CD31+ TDECs (arrows) in control tumors, but not in U87-E5 tumors. Areas in dotted boxes are magnified on the right. Scale bar: 50 μm