Fig 2: RBM39 promotes NSCLC cell stemness and EMT.(A–D) RBM39 is highly expressed in NSCLC. (A and B) RBM39 mRNA (A) and protein (B) levels are highly expressed in NSCLC through Gene Expression Profiling Interactive Analysis (GEPIA) and the University of Alabama at Birmingham Cancer Data Analysis Portal (UALCAN). (C) Survival analysis was conducted by dividing patients into two groups based on RBM39 expression levels. The high and low RBM39 expression groups were defined using the median expression value as the cutoff (low RBM39 = 519, high RBM39 = 519). These results suggest that high RBM39 expression is associated with a poorer prognosis in NSCLC. (D) Immunohistochemistry showed higher RBM39 expression in NSCLC tissues compared with para-carcinoma (n = 15). Bars = 50 μm. (E) RBM39 overexpression increases the expression of NSCLC-CSC surface markers and EMT-related markers. The protein levels of these markers in the cell lysate were measured by western blotting in A549 cells transfected with pLVX-IRES-Puro-RBM39 or pLVX-IRES-Puro-Vector (as a control). β-actin was used as a control. F RBM39 knockout reduces the expression of NSCLC-CSC surface markers and EMT-related markers. A549 cells with or without RBM39 knockout were analyzed by western blotting to detect the protein levels. (G and H) RBM39 promotes cell proliferation. (G) MTT (n = 3) was used to assess cell proliferation viability in A549 cells with RBM39 overexpression or knockout. (H) Colony formation ability of A549 cells with RBM39 overexpression or knockout was assessed using a colony formation assay. (I) Sphere formation assays (n = 3) were performed in indicated cell lines to detect the sphere-forming ability. RBM39 overexpression improves sphere-forming ability, and RBM39 knockout reduces sphere-forming ability. Representative images (I, left) and sphere number analysis (I, right) were shown. Bars = 200 μm. (J) The proportion of CD44+ CD133+ cells in each group was analyzed using flow cytometry. RBM39 overexpression enhances the number of CD133 and CD44 positive cells. RBM39 knockout reduces the number of CD133 and CD44 positive cells. Representative dot plots (J, left) and percentage of CD44+ CD133+ cells (J, right) were shown. (K) The effect of RBM39 on A549 motile ability was measured by scratch wound healing assay. RBM39 overexpression promotes A549 cell motility. RBM39 knockout inhibits A549 cell motility. Bars = 100 μm. (L) Transwell assays were performed to test the migratory and invasive potential of the A549 cells. RBM39 overexpression promotes A549 cell migratory and invasive, RBM39 knockout blocks A549 cell migratory and invasive. Bars = 200 μm. Data calculates the mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001. Statistical analysis was calculated using a two-tailed Student t test. The underlying data for Fig 2C, 2D, 2G, and 2I–2L can be found in S3 Data. NSCLC, non-small cell lung cancer; CSC, cancer stem cell; EMT, epithelial–mesenchymal transition; MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay; RBM39, RNA-binding protein 39.

Fig 3: Methylation of RBM39 reverses the inhibitory of Indisulam on NSCLC.(A) A549 and H460 cells transfected with empty vector, RBM39, or R92K, respectively, were treated with Indisulam, and the protein levels of stem-related markers were analyzed by western blotting. (B) MTT assays were performed to test the cell growth ability in the cells above treated with Indisulam. (C) The proportion of CD44+ CD133+ cells populations in indicated groups was quantified by flow cytometry. (D) Western blotting was used to detect N-cadherin, vimentin, and E-cadherin levels in A549 and H460 cells transfected with the indicated constructs and treated with Indisulam. (E) Wound healing assays were performed to evaluate the migratory ability of transfected cells after Indisulam exposure. Bars = 100 μm. (F) Transwell assays assessed the migratory and invasive capacities of the indicated cells above, Bars = 200 μm. Data calculates the mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001. Statistical analysis was calculated using the one-way ANOVA. The underlying data for Fig 6B, 6C, 6E, and 6F can be found in S3 Data. NSCLC, non–small cell lung cancer; RBM39, RNA-binding protein 39; MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.

Fig 4: Identification of hPDLSCs and macrophages. A hPDLSCs were acquired by the limited dilution method from periodontal ligament. a After crystal violet staining, cell clones scattered at the bottom of the large culture dish were visible to the naked eye after 14 days of culture. b Magnification of the cell clones in A(a), one clone was present here; scale bar = 200 μm. B hPDLSCs showed positive Alizarin Red staining; scale bar = 100 μm. C hPDLSCs showed positive Oil Red O staining; scale bar = 50 μm. D hPDLSCs positively expressed CD44 (98.6%), CD73 (99.2%), CD90 (97.0%), and negatively expressed CD34 (0.82%), CD45 (0.13%). E Vimentin staining for hPDLSCs was positive; scale bar = 100 μm. However, cytokeratin staining was negative for hPDLSCs; scale bar = 100 μm. F THP-1 grew in suspension manner; scale bar = 100 μm. G THP-1 was induced by PMA into M0-type macrophages and the cells grew adherently; scale bar = 100 μm. H The surface marker CD11b of M0-Mφ was identified