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. 2022 Nov 10;13:6808. doi: 10.1038/s41467-022-34617-7

Fig. 2. MiT/TFE nuclear localization and transcriptional activity is increased in vitro with TSC2 loss.

Fig. 2

Indirect immunofluorescence quantification for TFEB (a), and TFE3 (b) in TSC2 KO cells compared to WT controls (from experiments in Fig. S2a and S2b, respectively). n = 4 independent biological replicates, counting 2565 cells (for TFE3) and 780 cells (for TFEB), per replicate. p = 0.0369 for TFEB and p < 0.0001 for TFE3. c Indirect immunofluorescence for GFP in WT and TSC2 KO cells transiently transfected with TFEB-GFP for 24 h ± rapamycin (200 nM, 2 h) (Scale bar = 100 µm). d Quantification of nuclear/cytoplasmic TFEB-GFP fluorescence from experiments in c. n = 4 independent biological replicates, counting 150 cells per replicate. p = 0.0001 (WT vs TSC2 KO) and p = 0.0002 (TSC2 KO ± rapamycin). e TFEB and TFE3 expression in nuclear-fraction immunoblots of TSC2 KO cells compared to WT controls ± rapamycin (200 nM, 2 h) or torin (1 µM, 2 h). f, g Densitometry quantification of immune-blot experiments from e. n = 3 independent biological replicates. *p < 0.05, ***p < 0.001. h 4X-CLEAR luciferase activity in TSC2 KO cells compared to WT controls, normalized to Renilla luciferase. n = 4 independent biological replicates. p = 0.0282. i Representative TFEB and TFE3 immunohistochemistry images for TSC2 KO xenografts in NSG mice, compared to WT xenografts (Scale bar = 200 µm). j Digital quantification of mean nuclear TFE3 H-score from experiments in i. n = 6 (WT xenografts) and 8 (TSC2 KO xenografts) independent biological replicates. p < 0.0001. k In vivo growth and tumor volume of subcutaneous WT and TSC2 KO xenografts in NSG mice treated with vehicle, rapamycin or torin. n = 5 independent biological replicates. p = 0.0092 (rapamycin; WT xenografts) and p = 0.0005 (rapamycin; TSC2 KO xenografts). l Immunoblotting of tumor lysates from TSC2 KO xenografts (from experiments in k) treated with vehicle, rapamycin or torin (also see Fig. S3b). All graphs are presented as mean values ± SEM. Statistical analyses were performed using two-tailed Students t-test (panels a, b, h, and j) or one-way ANOVA with Dunnett’s test for multiple comparisons (panels d, f, and g) or Bonferroni’s test for multiple comparisons (panel k), if more than 2 groups. Source data are provided as a Source Data file.