Fig. 7
Exosomal transfer of lncSBF2-AS1 from chemoresistant cells to chemoresponsive cells and spread of TMZ resistance (a) The expression level of lncSBF2-AS1 in the culture medium of Rec GBM and N3T3rd cells treated with RNase (2 μg/ml) alone or combined with 0.1% Triton X-100 for 20 min. b Representative transmission electron microscopy (TEM) of exosomes secreted by TMZ-resistant cells. Scale bar, 100 nm. c The size distributions of exosomes derived from TMZ-resistant cells was analyzed by nanoparticle tracking assay. d Western blot analysis for exosomal marker CD63, CD81, and GM130 of exosomes derived from TMZ-resistant cells. Equal amount of these exosomes (500 ng) were used for Western blot analysis. e Exosomes were isolated from culture medium from Rec GBM and N3T3rd co-transfected with FAM-labelled SBF2-AS1 (green) and exosomal marker CD63 (red) and added to Pri GBM or N3S cell cultures. Confocal microscope was used to detect fluorescence signal in Pri GBM or N3S cells. Scale bar, 10 μm. f Western blot analysis of cleaved caspase-3 expression of exosome-treated chemoresponsive cells or untreated GBM cells in the presence of TMZ. Tubulin was used as the loading control. g TUNEL analysis of chemoresponsive GBM cells treated with TMZ-resistant exosomes in the presence of TMZ for 48 h. Data represents means of three independent experiments ± SEM. **P < 0.01, ***P < 0.001. Scale bar, 50 μm (h) Flow cytometry was used to measure the apoptosis of chemoresponsive GBM cells in the absence or presence of exosomes after exposure to 200 μM TMZ for 48 h. Data represents means of three independent experiments ± SEM. ***P < 0.001 (Rec GBM-exo: exosomes isolated from recurrent GBM cells; N3T3rd-exo: exosomes isolated from N3T3rd cells; Rec GBM-exo (shSBF2-AS1): exosomes isolated from shSBF2-AS1 recurrent GBM cells; N3T3rd-exo (shSBF2-AS1): exosomes isolated from shSBF2-AS1 N3T3rd cells)