Fig 2: The association between GPNMB expression and clinicopathological characteristics of 220 patients with glioma from the Chinese Glioma Genome Atlas dataset explored using the mRNA microarray data and clinical information. (A) Compared with non-tumor brain tissues, higher levels of GPNMB expression were observed in glioma tissues. (B and C) The GPNMB expression levels were compared in the (B) four gene expression-based and (C) four histological subtypes. (D) The levels of GPNMB expression in different WHO grades were compared. (E) Lower KPS were identified in patients with high GPNMB expression. (F) Kaplan-Meier analysis with the log-rank test demonstrated that high GPNMB expression was associated with shorter survival time compared with that of patients with low GPNMB expression. (G and H) Survival differences were compared between patients with high and low GPNMB expression in sub-populations aged (G) 10–30, (H) 31–50 and (I) 51–70 years. *P<0.05, **P<0.01 and ***P<0.001. GPNMB, glycoprotein non-metastatic melanoma protein B; KPS, Karnofsky performance scores.

Fig 3: Spearman correlation analysis between GPNMB and markers of angiogenesis, migration and invasion in the mRNA microarray dataset in Chinese Glioma Genome Atlas. (A-I) Correlations between GPNMB and (A) CD31, (B) ENG, (C) CXCR4, (D) TGFB1, (E) PLAU, (F) PLAUR, (G) MMP-2, (H) MMP-7 and (I) MMP-9. GPNMB, glycoprotein non-metastatic melanoma protein B; CD31, cluster of differentiation 31; ENG, endoglin; CXCR4, C-X-C motif chemokine receptor 4; TGFB1, transforming growth factor β1; PLAU, urokinase; PLAUR, PLAU receptor; MMP, matrix metalloproteinase.

Fig 4: Representative GPNMB staining in translocation RCC (tRCC) cases. Representative H&E and GPNMB IHC images for TFE3‐driven tRCC [PRCC–TFE3 and SFPQ–TFE3 cases confirmed by fluorescence in situ hybridization (FISH)] as well as a tRCC negative for cathepsin K immunostaining and a TFEB‐driven (FISH‐confirmed) tRCC case. All images were reduced from 200× magnification.

Fig 5: GPNMB is upregulated following TSC2 loss in a TFE3/TFEB‐ and mTORC1‐dependent fashion. (A) Immunoblotting of whole‐cell lysates from parental and TSC2 KO HEK293T cells for TSC2 and mTORC1 activation markers. (B) Immunoblotting of whole‐cell lysates from parental and TSC2 KO HEK293T cells for GPNMB. A non‐specific band (*) based on the siRNA experiment in supplementary material, Figure S2 is present in this cell line by immunoblotting. (C) Immunoblotting of cell lysates enriched for lysosomal content from parental and TSC2 KO HEK293T cells. (D) Immunoblotting of whole‐cell lysates from TSC2 KO HEK293T cells with or without 72‐h treatment with mTOR inhibitor rapamycin (200 nm). (E) RT‐qPCR for relative GPNMB gene expression in TSC2 KO HEK293T cells with or without genomic deletion of TFE3, TFEB, or TFEB and TFE3 (r = 3; ***p < 0.0001 by one‐way ANOVA). (F) Immunoblotting of whole‐cell lysates from TSC2 KO HEK293T cells with or without genomic deletion of TFE3, TFEB, or TFEB and TFE3. (G) Immunoblotting of whole‐cell lysates from TRI‐102 parental cells (TSC2 −/−) and TRI‐103 cells (TSC2 −/− with stable transfection of wild‐type TSC2) for TSC2 and mTORC1 activation markers. (H) RT‐qPCR for relative GPNMB gene expression in TRI‐102 and TRI‐103 cells. (I) Immunoblotting of cell lysates from TRI102 and TRI103 cells enriched for lysosomal content. (J) Immunoblotting of whole‐cell lysates from TRI‐102 cells with or without 72‐h treatment with mTOR kinase inhibitor Torin 1 (1 μm).