Fig 2: Activation of the PI3K-AKT pathway by the MAFB-NOTCH3 axis promotes osteosarcoma proliferation. (A) Dot plot of the KEGG enrichment pathways in the MMA treatment group. (B) The expression of components of the PI3K-AKT-mTOR pathway was confirmed by Western blot analysis. MNNG/HOS and U2OS cells were treated with 0, 5, 10, and 20 mmol/L MMA. (C) The expression of MAFB, NOTCH3, AKT, and p-AKT was detected by Western blot analysis. MNNG/HOS and U2OS cells treated with or without MMA were transfected with si-MAFB or si-NOTCH3, respectively. (D) The expression of MAFB, NOTCH3, AKT, and p-AKT was detected by Western blot analysis. MNNG/HOS and U2OS cells treated with or without MKK2206 were transfected with si-MAFB or si-NOTCH3, respectively. (E) The sensitivity of MNNG/HOS cells to MK2206 was detected in the CCK8 assay and measured in terms of the IC50 value. MNNG/HOS cells treated with MK2206 at different concentrations were transfected with si-NOTCH3 or si-control. Data were presented as mean ± SD of three replicates. The IC50 of the si-control group was 4.736 μg/mL. The IC50 of the si-NOTCH3 group was 0.1617 μg/mL. (F) Cell proliferation was detected in the colony-forming assay. Implanted MNNG/HOS cells were transfected with si-NOTCH3 or si-Control; cells were treated with MK2206 or PBS. (G) The cell migration potential was detected in the Transwell assay. The groups were formed as mentioned above. (H) Flow diagram for the xenograft assay in nude mice. (I) Results of the xenograft assay in nude mice. Implanted MMNNG/HOS cells were transfected with si-NOTCH3 or si-Control; cells were treated with MK2206 or PBS. n = 5. (J) The tumor tissue acquired from the nude mice xenograft assay was subjected to immunohistochemistry for Ki-67 staining. The groups were formed as mentioned above. n = 5.

Fig 3: MAFB promotes the transcription of NOTCH3 through protein-DNA interactions. (A) RNA sequencing analysis results shown in the heatmap indicate the differentially expressed genes in MNNG/HOS cells treated with or without MMA. (B) Downstream target genes of MAFB were predicted using the biological database network. (C) Wayne diagram showing differentially expressed genes identified by RNA sequencing and target genes of MAFB predicted using the Gene Transcription Regulation Database (GTRD). (D) mRNA expression of 11 overlapping genes based on the results of RNA sequencing. (E) The combination of MAFB protein and NOTCH3 chromatin was detected using the ChIP assay and evaluated by relative enrichment to input. Histone H3 served as the positive control. (F) MNNG/HOS and U2OS cells were infected with the indicated plasmids. After 48 h, the cells were harvested for RT-qPCR analysis. (G) The expression of NOTCH3 and MAFB was detected by Western blot analysis. MNNG/HOS or U2OS cells were transfected with Flag-MAFB or Flag-Control and si-MAFB or si-Control and treated with or without MMA, respectively.

Fig 4: MAFB up-regulation promotes the proliferation and migration of osteosarcoma cells. (A) Cell proliferation was detected in the colony-forming assay. MNNG/HOS and U2OS cells were transfected separately with si-Control or si-MAFB and treated with or without MMA. (B) Cell migration was detected in the Transwell assay. MNNG/HOS and U2OS cells were treated separately, as described above. (C) MAFB expression and subcellular co-localization were detected using immunocytochemistry and immunofluorescence assays, respectively. MNNG/HOS cells were treated separately, as described above. (D) Subcellular MAFB co-localization was analyzed using a scatter diagram. (E, F) The statistical graph of data from the colony-forming assay (E) and Transwell assay (F). Data were presented as mean ± SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001; n = 3. (G) Subcellular MAFB co-localization was measured by Pearson's r. The groups were formed as mentioned above. Data were presented as mean ± SD. ns, not significant; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001; n = 3. (H) Tumor tissue acquired from nude mice in the xenograft assay was used in the immunohistochemistry assay for Ki-67 staining. The groups were formed as mentioned above. Data were presented as mean ± SD of five replicates. ns, not significant; ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; n = 5. (I) Flow diagram for the xenograft assay in nude mice. (J) The result of the xenograft assay in nude mice. Implanted MNNG/HOS cells were transfected with si-MAFB or si-Control; MMA solution or PBS buffers were injected. n = 5. (K) The tumor tissue acquired from nude mice in the xenograft assay was used in the immunohistochemistry assay for Ki-67 staining. The groups were formed as mentioned above.

Fig 5: MAFB is overexpressed in osteosarcoma and its excessive up-regulation is induced by MMA. (A, B) RNA sequencing analysis results shown in the volcano plot (A) and heatmap (B) indicated the presence of 980 differentially expressed genes in MNNG/HOS cells treated with or without MMA. (C) Cell viability was determined using the CCK8 assay. MNNG/HOS cells were transfected with specific si-RNA and observed at 0, 12, 24, 36, and 48 h after MMA treatment. Data were presented as the mean ± standard deviation (SD) of three independent experiments. ns, not significant; ∗∗∗P < 0.001; n = 3. (D, E)MAFB expression in MNNG/HOS and U2OS cells was detected using Western blot analysis. Cells were treated with 0, 5, 10, or 20 mmol/L MMA and detected after 24 h (D). Cells were treated with 10 mmol/L MMA and detected at 0, 12, 24, and 48 h after treatment (E). (F)MAFB mRNA expression in sarcoma based on patient age determined with the TCGA data using the UALCAN web tool. Normal (n = 2), 21–40 years old (n = 22), 41–60 years old (n = 104), 61–80 years old (n = 111), and 81–100 years old (n = 20). (G)MAFB expression in one normal osteogenic cell line and four osteosarcoma cell lines was detected using Western blot analysis. (H)MAFB mRNA expression was detected by RT-qPCR analysis. Six pairs of T/ANT specimens from our clinical specimen bank were tested. “T" refers to tumor tissue and “ANT” refers to adjacent non-tumor tissue. Data were presented as the mean ± SD of three independent experiments. ns, not significant; ∗∗P < 0.01; ∗∗∗P < 0.001. (I, J)MAFB expression in clinical specimens was detected by Western blot analysis (I). Data were presented as the mean ± SD of three independent experiments (J). ns, not significant; ∗∗P < 0.01; ∗∗∗P < 0.001. (K) Immunohistochemistry results of MAFB staining in a part of the clinical specimens. (L) A two-sided paired Student's t-test was used to analyze the difference in the IHC staining rate of MAFB and serum MMA levels between the young group (age ≤25) and the old group (age ≥45). Data were presented as the mean ± SD of three independent experiments. ∗∗∗P < 0.001. (M) Correlation analysis between the serum concentration of MMA and the IHC staining rates of MAFB was drawn by a scatter diagram and measured using Pearson's r (r = 0.8811) and P value (P < 0.0001).