Fig 2: Low expression of RBM47 mRNA is associated with metastasis, nodal status, and poor survival in CRC patients. (a) Association of RBM47 mRNA expression with metastasis (right) and nodal status (left) in the TCGA COAD dataset. (b) Association of RBM47 mRNA expression with overall and relapse free survival in the TCGA COAD dataset. (c–g) Associations of RBM47 mRNA expression with relapse free survival in indicated CRC dataset. (h) Association of RBM47 mRNA expression with the consensus molecular subtypes (CMS) of colorectal cancer in TCGA and GSE39582 datasets.

Fig 3: Low expression of RBM47 protein is associated with metastasis and positive nodal status in CRC patients (a) Association of RBM47 protein expression with liver metastasis (M1) and nodal metastasis (N+) in the M0/M1 patient collection (N = 86). Left panel: representative IHC staining results of patient samples with staining scores. Right panel: quantification of IHC staining and association with metastasis (M0 – no metastases present, M1 – liver metastases present) and nodal status (N0 – no tumor cells present in lymph nodes, N+ - tumor cells present in lymph nodes). The scale bar in low magnification images (upper part) represents 100 μm and the bar in high magnification images (lower part) represents 50 μm. (b) Expression of RBM47 mRNA in normal colonic tissue and colon tumors in the TCGA COAD dataset. In the left panel expression of RBM47 in paired samples from the same patient are shown and in the right panel all samples are shown. (c) Exemplary IHC results showing expression of RBM47 protein in a colon cancer (red arrow) and adjacent normal colonic mucosa (green arrow). The scale bar represents 100 μm. (d) Immunoblot analysis of RBM47 expression in a cell line panel consisting of epithelial-like (E) and mesenchymal-like (M) colorectal (CRC), breast (BC), and prostate (PC) cancer cell lines.

Fig 4: Inhibition of RBM47 induces metastasis. Formation of lung metastases after tail-vein injection of DLD-1–Luc2 cells transfected with control or RBM47-specific siRNA. (a) Representative images of luciferase signals after D-luciferin injection at the indicated time points after xenografting. (b) Weekly measurements of total photon flux. Results are the mean ± SD (n = 5). **P < 0.01. (c) Representative lungs 8 weeks after tail vein injection. (d) H&E staining of lung tissue. Scale bars represent 500 μm. (e) Number of metastatic nodules in lungs 8 weeks after tail vein injection of the indicated DLD1 cells into mice (n = 5). (f) Model of regulation of RBM47 expression and its downstream targets, which presumably mediate its effects on EMT and cancer progression.

Fig 5: Inhibition of RBM47 induces EMT, cell migration and invasion. (a) Phase contrast microscopy of the indicated CRC cell lines transfected with control or RBM47-specific siRNA. Scale bars represent 100 μm. (b) Western blot analysis of the indicated proteins in CRC cell lines transfected with control or RBM47-specific siRNA for 72 h. (c) qPCR analysis of the indicated mRNAs in the indicated CRC cell lines transfected with control or RBM47-specific siRNA for 72 h. (d) Wound healing assay of the indicated CRC cell lines transfected with control or RBM47-specific siRNA 48 hours before a scratch was generated. (Upper panels) Representative photographs of the initial wound area and the same area 24 hours later. (Lower panels) Quantification of wound closures: The width of scratches in 2 independent wells was analyzed for each state. Results represent the average (%) of wound closure. (e) Relative invasion of indicated CRC cells in matrigel-coated Boyden chambers transfected with control or RBM47-specific siRNA for 48 hours. In c, d, and e mean values ± SD (n = 3) are provided. *P < 0.05; **P < 0.01; *** P < 0.001.