Fig 2: . RBM47 interacts with NONO to promote the phenotype of MB cellsA. Expression correlations between RBM47 and NONO in MB samples. Data were collected from R2 database. B. Detection of RBM47 Binding to NONO by Caulmers Brilliant Blue Staining. C. RBM47 and NONO interact in 293FT cells, DAOY and ONS76 cells. D. E. Detection of the functional domain of RBM47 required for its interaction with NONO. Schematic diagram of truncated construct of NONO. HEK-293FT cells were co-transfected with Flag-NONO truncations and HA-RBM47 for 48 h before Co-IP assay. F. RBM47 and NONO co-localize in the nucleus. Immunofluorescence analysis in MB cells using anti-RBM47 and NONO antibodies. Scale bar = 5 μm. G. IHC assay for detecting the expression of NONO after knockdown RBM47. H.I. Detection of NONO expression after knockdown RBM47 by Western blot and qRT-PCR assays. J. Western blot analysis of the turnover of NONO. K. Western blot analysis of control and RBM47-knockdown DAOY and ONS76 cells treated with MG132. L. In the presence of MG132, 293FT cells were co-transfected with Flag-NONO, shRBM47 and HA-UB plasmids, then the ubiquitination level of NONO was detected by co-IP. All data were expressed as the mean ± SD, n = 3. Student’s t-test was performed to analyze significance. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 3: The HDAC2/RBM47/NONO axis regulates P13K-AKT signalingA. Western blot analysis of PI3K-AKT signaling protein expression after HDAC2 knockdown in MB cells. B. Western blot analysis of PI3K-AKT signaling pathway protein expression in MB cells after treatment with HDAC2 inhibitors C. Western blot analysis of PI3K-AKT signaling protein expression after RBM47 knockdown in MB cells. D. Western blot analysis of PI3K-AKT signaling protein expression after NONO knockdown in MB cells. All data were expressed as the mean ± SD, n = 3. Student’s t-test was performed to analyze significance. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 4: HDAC2 promotes RBM47 expression though H3K27 deacetylation in MB cellsA. Volcano plot showing fold change (log2) in MB cell based on mRNA expression levels for HDAC2 high versus ground expression. Data from GEO database (GSE85217). B.C. GO enrichment analysis of HDAC2 gene high expression versus low expression in MB cells. D. GSEA enrichment map of mRNA binding signaling pathway in MB cells with high HDAC2 expression versus ground expression. E. RNA binding to merged mRNAs of related genes and in our differential gene. F. Heatmap of correlation between related genes and HDAC2. G. Expression correlations between HDAC2 and RBM47 in MB samples. Data were collected from R2 database. H. I. Protein and mRNA levels of RBM47 in HDAC2-knockdown cells were examined via Western blot and qRT-PCR. J. After suppressing HDAC2 expression using HDAC2 inhibitors, the protein levels of RBM47 were detected in DAOY cells using Western blotting. K. H3K27ac modification in RBM47 gene promotor using the Cistrome database. L. Dual luciferase assay for detecting the activity of RBM47 promoter in MB cells with knockdown/overexpression of HDAC2. M. ChIP-qPCR assays determine the binding area. N. Detection of acetylated histone H2AK5 and H3K27 in MB WT or knockdown HDAC2 cells. O. IHC assay for detecting the expression of RBM47 after knocking down HDAC2 in vivo.All data were expressed as the mean ± SD, n = 3. Student’s t-test was performed to analyze significance. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 5: The HDAC2/RBM47/NONO axis regulates P13K-AKT signalingA. The proliferation and invasion of cells under overexpression HDAC2 after RBM47 knockdown was determined via MTT. B.C. MTT assay was performed to test the effect of HDAC2/RBM47 restoration on cell proliferation. D.E. Representative fluorescent images and quantification of EdU staining of MB cells expressing HDAC2 knockdown or restoration of HDAC2/RBM47. Scale bar, 20 μm. F. Transwell assays were performed to test the effect of HDAC2/RBM47 restoration on MB cells to detect invasion ability. Scale bar, 20 μm. G. IHC assays detects changes in Ki67 after restoration of HDAC2/RBM47 expression. H.I. In vivo analyses of size and volume of CDX tumors that were hypodermically injected with treated DAOY cells. J. Western blot assays detects changes in related proteins. K.L. MTT assay was performed to test the ability to restore the proliferation of NONO and NONO structural domain Δ1/2 MB cells after knockdown of RBM47. N.O. EDU assay was performed to test the ability to restore the proliferation of NONO and NONO structural domain Δ1/2 MB cells after knockdown of RBM47. M. Western blot analysis of restoration of NONO expression after knockdown of RBM47 in MB cells with expression of proteins associated with value-added after Δ1/2 of the NONO structural domain.All data were expressed as the mean ± SD, n = 3. Student’s t-test was performed to analyze significance. *P < 0.05, **P < 0.01, ***P < 0.001.