Fig 2: Importance of the serine residue in epithelial-mesenchymal transition induction and cell migration of glycoprotein non–metastatic melanoma protein B (GPNMB) protein. A,B, mRNA expression levels of Cdh1 (A) and fibronectin (B), in NMuMG-mock, NMuMG-GPNMB(WT) (G3 and G8), and NMuMG-GPNMB(SA) (SA8 and SA10) cells were measured by quantitative PCR (qPCR) and normalized to those levels of ß-actin. Data are presented as mean ± SD; n = 3. ****P < .0001, one-way ANOVA with Tukey’s multiple comparison test. C, Immunoblotting for E-cadherin and fibronectin in NMuMG-mock, NMuMG-GPNMB(WT) (G3 and G8), and GPNMB(SA) (SA8 and SA10) cells was performed. ß-actin was used as a loading control. D, Immunofluorescence staining of E-cadherin (green) in NMuMG-mock, NMuMG-GPNMB(WT) (G3 and G8), and NMuMG-GPNMB(SA) (SA8 and SA10) cells. Nuclei were counterstained with propidium iodide (red). Scale bar, 20 µm. E, Migration of NMuMG-mock, NMuMG-GPNMB(WT) (G3), and NMuMG-GPNMB(SA) (SA8 and SA10) cells was examined by Transwell migration assay. Data are presented as mean ± SD, n = 3 replicates, representative of three independent experiments. ****P < .0001, one-way ANOVA with Tukey’s multiple comparison test. Scale bar, 100 µm. F-H, mRNA expression levels of Snail (F), Slug (G), and Zeb1 (H) in NMuMG-mock, NMuMG-GPNMB(WT) (G3 and G8), and NMuMG-GPNMB(SA) (SA8 and SA10) cells were analyzed by qPCR and normalized to those levels of ß-actin. Data are presented as mean ± SD; n = 3. **P < .01, ***P < .001, ****P < .0001, one-way ANOVA with Tukey’s multiple comparison test. Data are representative of three independent replicates

Fig 3: High level of GPNMB expression in the renal tumors from BHD patients and Flcn heterozygous knockout mice.(A) GPNMB mRNA and (B) protein expressions were measured by quantitative RT-PCR and Western blotting, respectively in the renal tumors and normal kidneys. (C) Immunohistochemical staining of GPNMB in the tumors adjacent to normal kidneys from BHD patients or Flcn-heterozygous knockout mice. GPNMB staining in the UOK257 xenograft tumors grown in nude mice.

Fig 4: HE, EGFR, GPNMB, and VEGF staining of the 3 types of lymph nodes. EGFR, epidermal growth factor receptor; GPNMB, glycoprotein nonmetastatic melanoma protein B; HE, hematoxylin and eosin; VEGF, vascular endothelial growth factor.

Fig 5: Proliferative NHEKs express more GPNMB than differentiated NHEKs. NHEKs were cultured in the presence of high concentrations of calcium for 24 hrs. (a) The dose-dependent effect of calcium on the expression of GPNMB mRNA was measured by real-time PCR. The data were expressed as mean ± SD (n = 3). *P < 0.05 and **P < 0.01 vs control (Student’s t-test). (b) Total protein was extracted from the cell lysates, treated with PNGase, and subjected to western blot analysis. The protein levels of GPNMB were quantified relative to those of ß-actin. The data were expressed as mean ± SD (n = 3). ***P < 0.001 vs control (Student’s t-test). The full-length blot has been presented in Supplementary Fig. S10.