Fig 1: RREB1 binds to the promoter region of MELTF-AS1 and activates its transcription in MELTF-AS1(A and B) qRT-PCR analysis of MELTF-AS1 expression in 143B and MG63 cells following RREB1 knockdown and overexpression. (C) qRT-PCR analysis of RREB1 expression in 40 pairs osteosarcoma tissues and corresponding adjacent normal tissues. (D) In situ hybridization (ISH) analysis of RREB1 expression in 40 pairs osteosarcoma tissues and corresponding adjacent normal tissues. (E) ChIP-qRT-PCR analysis of RREB1 occupancy in the MELTF-AS1 promoter in 143B and MG63 cells. (F) Luciferase reporter assays of the 143B cells transfected with reporter vector containing full length of the MELTF-AS1 promoter or containing the promoter with deletion of binding sites, the pcDNA-RREB1 vector, or an empty vector. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.
Fig 2: Schematic representation of the potential molecular mechanisms of MELTF-AS1 involved in osteosarcomaMechanistically, RREB1 activates MELTF-AS1 transcription in osteosarcoma. MELTF-AS1 competes with MMP14, a key pro-metastasis gene, for miR-485-5p and relieves the inhibitory effect of miR-485-5p on MMP14, thereby leading to increased MMP14 expression and metastasis ability of osteosarcoma cells.
Fig 3: RREB1 functions as a transcription factor for Foxo1, and NICD2 interacts with RREB1. (A) Bioinformatic prediction of RREB1 binding sites within the Foxo1 promoter region. (B) Correlation between RREB1 and Foxo1 expression levels in lymphocyte populations. (C) Schematic diagram of four predicted RREB1 binding sites (P1–P4) on the Foxo1 promoter. (D) ChIP–PCR validation of RREB1 enrichment at the Foxo1 P2 site. (E, F) Luciferase reporter assays assessing RREB1‐dependent Foxo1 promoter activity. (G) Immunofluorescence staining showing nuclear colocalization of NICD2 and RREB1. (H) Coimmunoprecipitation (co‐IP) analysis of the NICD2‐RREB1 protein interaction. (I) Results from the results of the GST pull‐down assay confirm direct binding between NICD2 and RREB1. Data are presented as the mean ± SD from three independent experiments; n = 3; ns, not significant (p > 0.05).
Fig 4: NICD2 modulates the subcellular distribution of RREB1 in MoT cells. (A) Immunofluorescence analysis of RREB1 subcellular localization under different Notch2 signaling conditions. (B, C) Western blot quantification of RREB1 protein levels in cytoplasmic (B) and nuclear (C) fractions. (D) Total RREB1 expression in shNICD2 and oeNICD2 MoT cells. Data are presented as bar graphs with individual data points; n = 3.
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