Fig 2: GPNMB is induced and secreted in UHRF1-lacking 3T3-L1 preadipocytes. (A) Venn diagram showing 846 common upregulated genes between preadipocyte and adipocyte data sets. (B) The Expression log ratio of GPNMB in both datasets. (C, D) Relative mRNA expression of GPNMB in NT, UHRF1 KO G1, shScramble, and shUHRF1 C2 at different time-points during adipocyte differentiation by qPCR. (E, F) GPNMB protein levels in NT, UHRF1 KO G1, shScramble, and shUHRF1 C2 by Western blot analysis with Actin as the loading control. (G, H) ImageJ software-based Quantitation of GPNMB protein in Western blot results by normalizing to Actin. Fold change values were calculated relative to the D0 control. (I) Immunostaining of GPNMB in NT, UHRF1 KO G1 preadipocytes where nuclei are stained in blue (DAPI), GPNMB stained in green (FITC). Images were taken at 63 × magnification. (J) Quantification of GPNMB fluorescence intensity in NT and UHRF1 KO G1 preadipocytes immunostaining by ImageJ software. (K) ELISA detected the secreted level of GPNMB in the conditioned media of NT and UHRF1 KO G1 preadipocytes. (L, M) Western blot analysis of GPNMB and its quantification graph in the conditioned media of NT and UHRF1 KO G1 preadipocytes. Data are mean ± SEM of 3 independent experiments. Statistical analyses were performed using ordinary one-way ANOVA, *P < 0.05.

Fig 3: Renal tumors from A/J Tsc2 +/− mice show increased GPNMB expression. (A) Hematoxylin and eosin (H&E) and TSC2, p‐S6, and p‐4EBP1 IHC staining of cystadenoma lesions occurring in 14‐month‐old A/J Tsc2 +/− mice. Scale bar: 500 μm. (B) Laser capture microdissection (LCM) of renal cystadenomas from 14‐month‐old A/J Tsc2 +/− mice guided by p‐S6 immunostaining for mTORC1 activation. Images reduced from 100× magnification. Scale bar: 250 μm. (C) RT‐qPCR for relative GPNMB gene expression in normal kidney parenchyma and LCM‐captured renal cystadenoma tissue. (D) H&E and GPNMB IHC staining of oncocytic vacuolated carcinomas occurring in 14‐month‐old A/J Tsc2 +/− mice. Scale bar: 500 μm. Right panels are higher‐magnification views of the outlined area in each image.

Fig 4: Representative GPNMB immunohistochemical (IHC) staining in TSC1/2/mTOR‐related tumors. (A) Representative H&E and GPNMB IHC images for eosinophilic solid and cystic carcinoma (ESC), eosinophilic vacuolated tumor (EVT), and low‐grade oncocytic tumor (LOT). All images were reduced from 200× magnification. (B) Representative H&E and GPNMB IHC images for conventional angiomyolipoma (AML) and perivascular epithelioid cell tumor (PEComa). 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).