Fig 2: PRPS1 promotes melanoma cells proliferation in vivo. (A–C) BALB/c nude mice were injected with SK-MEL-110 cell that were stably transfected with PRPS1 overexpression and the control. Representative images of mice with control (top) and PRPS1-overexpressing (lower) xenograft tumors (A). The tumor volume (B) and body weight (C) were measured. (n=6/group) (D–F) BALB/c nude mice were injected with A875 cell that were stably transfected with PRPS1 knockdown and the control. Representative photographs of mice with control (top) and PRPS1- knockdown (lower) xenograft tumors (D). The tumor volume (E) and body weight (F) were measured. (n=6/group) (G–J) The protein level of PRPS1 (G, H) and the cell cycle related protein (I, J) levels of tumors in each xenograft tumors group were measured by western blot analysis. The data represent three independent experiments. The data related to tumor volume were statistically analyzed by two-way ANOVA, and the other data were analyzed by unpaired-sample Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 3: PRPS1 advances the migration and invasion of melanoma cells. (A) Representative scratch-wound images and the data analysis of PRPS1 over-expression and knock-down melanoma A875 and SK-MEL-110 cells 0h and 24h after scratch. (B) Representative images of transwell migration of stably transformed melanoma cells after 24h of starvation (top panel). Quantification of the number of migrating cells per field (bottom panel). (C) Representative images of transwell invasion assay pictured 24h (top panel). Quantification of the number of invasion cells per field (bottom panel). (D) The expression of EMT-associated proteins in the A875 and SK-MEL-110 melanoma cells with PRPS1 overexpression or knock-down and the control cells. The data represent three independent experiments. (*p<0.05, **p<0.01, *** p<0.001, ns mean no significant difference).

Fig 4: NRF2 is a transcription factor of PRPS1-mediated malignant progression of melanoma. (A) Consensus binding sites for NRF2 on the PRPS1 promoter was analyzed by JASPAR database. (B) ChIP assay was performed to assess NRF2 in the PRPS1 promoter region in A875 and SK-MEL-110 cells. (C) 293T cells were transfected with corresponding plasmids for 48h. Luciferase dual reporter assays were performed to measure the luciferase activity of PRPS1-luc. (D) NRF2 expression in nuclei and cytoplasm of A875 and SK-MEL-110 cells overexpressing or knockdown PRPS1. (E) Proposed model of the relationship between PRPS1 and NRF2 in melanoma cells. The data represent three independent experiments. The data represent three independent experiments. Each bar represents mean ± SD. p values were calculated using a student t-test (*p < 0.05, **p < 0.01, ***p < 0.001 vs. the control).

Fig 5: NRF2 is positively correlated with PRPS1 expression in melanoma. (A) Spearman correlation analysis of PRPS1 and NRF2 mRNA expression levels was performed in melanoma and normal skin tissues through GEPIA website. (B, C) The mRNA (B) and protein (C) level of PRPS1 were analyzed in the stably NRF2 overexpression or knockdown A875 and SK-MEL-110 and that the control cells. (D–F) The stably PRPS1 overexpression A875 and the control cells were treated with TP-155 (NRF2 activator) for 24h. The stably PRPS1 knockdown SK-MEL-110 and the control cells were incubated with ML385 (NRF2 inhibitor) for 24h. The expression of PRPS1 was detected by using western blot in each group cells (D). The cell proliferation rate was detected by MTS assay (E). The rate of migration was measured by transwell assay (F). TP-155 (+): 0.05uM, TP-155 (++): 0.1uM. ML385 (+): 2.5uM, ML385 (++): 5uM. The cell migration ability was evaluated by transwell migration (F). TP-155 (+): 10nM, TP-155 (++): 25nM. ML385 (+): 1uM, ML385 (++): 2uM. (G, H) The expression of NRF2 in the xenograft tumors (G) and the lung metastases of nude mice (H) were detected by western blot analysis. Statistical analysis was carried out with unpaired-sample Student’s t test or two-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ns mean no significant difference.