Fig. 6: Constitutive active TrkB and the SQSTM1-NTRK2 oncogene reduce migratory activity of U87MG cells. | Cancer Gene Therapy

Fig. 6: Constitutive active TrkB and the SQSTM1-NTRK2 oncogene reduce migratory activity of U87MG cells.

From: Atypical cellular responses mediated by intracellular constitutive active TrkB (NTRK2) kinase domains and a solely intracellular NTRK2-fusion oncogene

Fig. 6

A Outline: inducible lentiviral expression vector. B Western blotting of whole-cell lysates generated from U87MG cells expressing the TrkB kinase construct. Cells transduced with the indicated lentiviral constructs were maintained as a polyclonal cell line, and puromycin was used to select transduced cells. In the absence of doxycycline, cells did not express the corresponding proteins. Doxycycline was added to induce Trk construct expression for 48 h. Constitutive activation of Trk was verified using pTrk-kin. γ−Adaptin was used as a loading control. C Representative phase-contrast microscopy images of the migration assay. U87MG cells stably expressing either TrkB-wt, TrkB-YFF, or the fusion protein SQSTM1-NTRK2 via the Tet-on system. The image in the upper left corner shows the cells in the culture after removing the silicone insert (t = 0). The other images represent the situation 24 h later. Note the morphological changes in TrkB-wt and SQSTM1-NTRK2 expressing cells versus TrkB-YFF. D Migratory activity of U87MG cells expressing the indicated Trk kinase constructs. The relative numbers of cells relative to the initial cell-free gap are shown. Cell counts were normalized to the mean number of TrkB-YFF-expressing cells. Migratory activity is shown relative to TrkB-YFF, which expresses the same structural protein domains as TrkB-wt but is mutated at Y705 and Y706 (see also S8); n = 4 biological replicates. E Immunostaining of U87MG cells expressing inducible TrkB-wt, TrkB-YFF, or the NTRK fusion construct SQSTM1-NTRK2. Immunofluorescence of the TrkB receptor domain (green), pTrk-kin (magenta), and Acti-stain-670 phalloidin (phall, cyan). Yellow arrows indicate constitutive pTrk close to F-actin. TrkB-YFF-expressing cells were labeled with anti-pTrk-PLCγ because the antibody-binding site of anti-pTrk-kin was mutated in this construct. SQSTM1-NTRK2 does not have a receptor domain, as indicated. Confocal z-stack images; scale bar: 50 µm.

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