Fig. 3: TFE3-lysosome pathway enhances the tolerance of GBM therapy. | Nature Communications

Fig. 3: TFE3-lysosome pathway enhances the tolerance of GBM therapy.

From: Lysine-arginine imbalance overcomes therapeutic tolerance governed by the transcription factor E3-lysosome axis in glioblastoma

Fig. 3

A The representative images of TFE3 protein levels in sorted CD44-high and CD44-low TGS04 cells by immunocytochemistry and quantification of mean fluorescence intensity of nuclear TFE3 (CD44-high n = 87; CD44-low n = 89). Scale bars, 10 µm. B The representative images and quantification of the fluorescence intensity of DQ-BSA associated with lysosomal proteolytic activity (Control n = 67, TFE3-KO n = 64; Control n = 65, TFE3-OE n = 67). Scale bars, 10 µm. C The representative images and quantification of galectin-3 punctation in TFE3-WT and TFE3-OE TGS04 cells after treatment with LLOMe (50 µM) for 15 min (n = 3 independent samples). Scale bars, 10 µm. D, E Number and size of spheres formed by TFE3-WT and TFE3-KO TGS04 cells (D), or TFE3-WT and TFE3-OE KGS22 cells (E) (n = 3 technical replicates). F The effect of TFE3-OE on the sensitivity of TGS04 to TMZ was examined by the cell viability assay (n = 3 technical replicates). G Flow cytometric analyses of TFE3 and CD44 levels in which TGS04 cells expressing TFE3-EGFP. H Detection of lysosomal proteolytic activity for TFE3-EGFP-low (lowest 15% of total) and TFE3-EGFP-high (highest 15% of total) TGS04 cells. I Evaluation of mitochondrial ROS in indicated subpopulations of TGS04 cells. J The ratio of the CD44-high/TFE3-EGFP-high TGS04 cells to total cells after treatment of TMZ for 5 days. K–M Mitochondrial membrane potential (K), mitochondrial ROS (L), and oxygen consumption rate (OCR) (M) were detected in TGS04 cells after treatment of 5 nM bafA1 for 16–24 h. (n = 3 independent samples, data are presented as the means ± SEM for OCR). Data are presented as the means ± SD. For the boxplots (D, E), the line inside the box shows the median value. The bounds of the box represent the 25th–75th percentiles, with whiskers at minimum and maximum values. Statistical comparison (A, B, E) using unpaired two-tailed Student’s t-tests. Statistical comparison (C, D) using a one-way ANOVA. Statistical comparison (F) using a two-way ANOVA. ns, not significant, ****p < 0.0001. Experiments were repeated at least three times with similar results. Source data are provided with this paper.

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