Figures and data
Expression of RBMX2 after infection, and RBMX2 did not affect cell proliferation but inhibited cell survival during M. bovis infection.
(A, B) The expression of RBMX2 in EBL cells infected by (A) M. bovis and (B) M. bovis BCG were analyzed by RT-qPCR. Data were represented by fold expression relative to uninfected cells. (C,-E) The expression of RBMX2 mRNA in (E) BoMac cells, (F) A549 cells, and (G) bovine lung alveolar primary cells infected by M. bovis were analyzed via RT-qPCR. Data were represented by fold expression relative to uninfected cells. (F) The expression of RBMX2 mRNA in clinical TB tissues infected by M. bovis via RT-qPCR. Data were represented by fold expression relative to uninfected tissues. (G, H) The effect of RBMX2 on the proliferation of EBL cells was observed by EDU assay. Data were represented by the Fluorescence cells relative to WT EBL cells. (I) The effect of RBMX2 on the proliferation of EBL cells was observed by CCK-8 assay. Data were represented by the absorbance value relative to WT EBL cells. (J) Detection of the ability of RBMX2 knockout Polyclonal EBL cells resisting to M. bovis infection by CCK-8 assay. Data were represented by the absorbance value relative to WT EBL cells after M. bovis infection. (K) Detection of the ability of different RBMX2 knockout site Monoclonal EBL cells against M. bovis infection by CCK-8 assay. Data were represented by the absorbance value relative to WT EBL cells after M. bovis infection. (L) Detection of the ability of RBMX2 slicing H1299 cells against M. bovis infection by CCK-8 assay. Data were represented by the absorbance value relative to Sh-NC H1299 cells after M. bovis infection. One-way ANOVA and two-way ANOVA were used to determine the statistical significance of differences between different groups. Ns presents no significance, * presents p < 0.05, **presents p < 0.01, and *** presents p < 0.001 indicate statistically significant differences. Data were representative of at least three independent experiments.
Transcriptome and proteomic analysis in RBMX2 knockout and WT EBL cells after M. bovis infection.
(A) The heat map illustrates some genes that had been all enriched in RBMX2 knockout and WT EBL cells after M. bovis infection in 0, 24, and 48hpi. Red represents upregulated genes, and blue represents downregulated genes. Each group represented three independent samples. (B) GO Analysis of all enriched genes in 0, 24 and 48hpi. Data were represented as all enriched pathways in RBMX2 knockout EBL cells relative to WT EBL cells after M. bovis infection thrice. (C) KEGG Analysis of all enriched genes in 0, 24, and 48hpi. Data were represented as all enriched pathways in RBMX2 knockout EBL cells relative to WT EBL cells after M. bovis infection in three times. (D) GO Analysis of all enriched proteins in 48hpi. Data were represented as all enriched pathways in RBMX2 knockout EBL cells relative to WT EBL cells after M. bovis infection. (E) KEGG Analysis of all enriched genes in 48hpi. Data were represented as all enriched pathways in RBMX2 knockout EBL cells relative to WT EBL cells after M. bovis-infection (MOI 20) in three times. (F) Identification the expression of related genes mRNA enriched by RT-qPCR. Data were represented as the fold expression in RBMX2 knockout EBL cells relative to WT EBL cells after M. bovis infection. Two-way ANOVA was used to determine the statistical significance of differences between different groups. Ns presents no significance, * presents p < 0.05, **presents p < 0.01, and *** presents p < 0.001 indicate statistically significant differences.
RBMX2 had the potential to induce the disruption of tight junctions in EBL cells after M. bovis infection.
(A, B) KEGG analysis was conducted to identify the (A) down-regulated and (B) up-regulated pathways among the enriched genes after M. bovis infection of WT EBL cells. Data were relative to WT EBL cells without M. bovis infection. (C, D) The expression of epithelial cells tight junction-related (C) mRNAs (TJP1, CLDN-5, CLDN-7, and OCLN) and (D) proteins (ZO-1, CLDN-5, and OCLN) were assessed after M. bovis infection of WT EBL cells via RT-qPCR and WB. Data were relative to WT EBL cells without M. bovis infection. (E) Cell adhesion ratio was evaluated via cell adhesion assay after WT EBL cells were infected with M. bovis using High-content imaging. Data were relative to WT EBL cells without M. bovis infection. Scale bar: 20 μm. (F, G) The expression of epithelial tight junction-related (F) mRNAs (TJP1, CLDN-5, and OCLN) and (G) proteins (ZO-1, CLDN-5, and OCLN) in RBMX2 knockout EBL cells after M. bovis-infection through RT-qPCR and WB. Data were relative to WT EBL cells with M. bovis infection. (H) Cell adhesion assay was conducted to assess the cell adhesion ratio of RBMX2 knockout EBL cells after infection with M. bovis. Data were relative to WT EBL cells with M. bovis infection. Scale bar: 20 μm. (I) Expression of inflammatory factors-related factors (IL-6, IL-1β, and TNF) were assessed after RBMX2 knockout EBL cells infected by M. bovis. Data were relative to WT EBL cells with M. bovis infection. T-test and Two-way ANOVA were used to determine the statistical significance of differences between different groups. Ns presents no significance, * presents p < 0.05, **presents p < 0.01, and *** presents p < 0.001 indicate statistically significant differences. Data were representative of at least three independent experiments.
RBMX2 facilitated the disruption of epithelial tight junctions through the promotion of p65 protein phosphorylation and translocation and then enhanced the processes of M. bovis adhesion, invasion, and intracellular survival.
(A, B) Activation of the MAPK pathway-related protein and p65 protein were activated after RBMX2 knockout and WT EBL cells infected by M. bovis via WB. Data were relative to WT EBL cells with M. bovis infection. (C, D) Expression of tight junction-related proteins (ZO-1, CLDN-5, and OCLN) was assessed in RBMX2 knockout EBL cells treated with three p38/p65/JNK pathways activators after M. bovis infection via WB. Data were relative to RBMX2 knockout EBL cells untreated activators with M. bovis infection. (E, F) Evaluate the impact of three p38/p65/JNK pathways activators on the ratio of intercellular adhesion via cell adhesion assay. Data were relative to RBMX2 knockout EBL cells untreated activators with M. bovis infection. Scale bar: 20 μm. (G, H) Evaluate the silencing efficiency of siRNA on p65 protein expression and its impact on the expression of ZO-1, CLDN-5, and OCLN proteins through WB. Data were relative to SiRNA-NC in WT EBL cells with M. bovis infection. (I) Detect the correlation between p65 expression and ZO-1 in WT EBL cells after M. bovis infection via IF analysis. ZO-1 is stained with green fluorescence, p65 is stained with yellow fluorescence, and the nucleus is stained with blue fluorescence. Data were relative to SiRNA-NC in WT EBL cells with M. bovis infection. (J) The effect of p65 silencing on the invasive ability of M. bovis in WT EBL cells. Data were relative to SiRNA-NC in WT EBL cells with M. bovis infection. (K) The effect of RBMX2 on the nuclear translocation of p65 protein after M. bovis infection using WB. β-actin presents cytosol and Lamin A/C presents nucleus. Data were relative to RBMX2 knockout EBL cells after M. bovis infection. (L) The effect of RBMX2 on the nuclear translocation of p65 protein after BCG-infection using High-content real-time imaging. Using pCMV-EGFP-p65 plasmid transfect RBMX2 knockout and WT EBL cells. The nucleus is stained with blue fluorescence. Data were relative to WT EBL cells without BCG infection. (M, N, O) The impact of M. bovis on the adhesion, invasion, and intracellular survival of RBMX2 knockout and WT EBL cells through plate counting. Data were relative to WT EBL cells after M. bovis infection. (P, Q, R) The impact of M. bovis on the adhesion, invasion, and intracellular survival of Sh-NC and Sh-RBMX2 H1299 cells through plate counting. Data were relative to H1299 ShNC cells after M. bovis infection. One-way ANOVA and two-way ANOVA were used to determine the statistical significance of differences between different groups. Ns presents no significance, * presents p < 0.05, **presents p < 0.01, and *** presents p < 0.001 indicate statistically significant differences. Data were representative of at least three independent experiments.
RBMX2 is highly expressed in tumor tissues and regulates cancer-related metabolites
(A) A comparative analysis of the functional domains of the RBMX2 protein across ten different species. (B) Analyzing the expression patterns of RBMX2 in pan cancer using TIMER2.0 cancer database. (C) The expression of RBMX2 in different lung cancer cells and normal lung epithelialcells via RT-qPCR. Data were relative to normal lung epithelialcells (BEAS-2B). (D-E) The expression of RBMX2 in lung cancer clinical tissues via IF. RBMX2 is stained with yellow fluorescence, and the nucleus is stained with blue fluorescence. Data were relative to pericancerous lung tissues. Scale Bar: 5000 μm. (F) The expression of RBMX2 and p65 in LUAD and LUSC clinical tissues via IF. RBMX2 is stained with red fluorescence, p65 is stained with green fluorescence, and the nucleus is stained with blue fluorescence. Data were relative to normal lung tissues. Scale Bar: 100 μm. (G) KEGG analysis of differential metabolite enrichment pathways in RBMX2 knockout EBL cells compared to WT EBL cells after M. bovis infection. (H) Dynamic distribution map of top 20 differential metabolites in RBMX2 knockout EBL cells compared to WT EBL cells after M. bovis infection.
RBMX2 facilitates EMT process in EBL cells after M. bovis-infected BoMac cells.
(A) A pattern diagram illustrated a M. bovis-infected BoMac cells inducing EMT of EBL cells coculture model, drawing by BioRender. (B) Detection of IL-6 and TNF expression levels in EBL cells and BoMac cells infected with M. bovis using RT-qPCR. Data were relative to BoMac cells without infection of M. bovis. (C) Detection of RBMX2 expression levels in a coculture model EBL cells after M. bovis infection using RT-qPCR. Data were relative to BoMac cells without infection of M. bovis. (D) Observation of morphological changes in EBL cells infected with M. bovis under electron microscopy. (E) EMT-related proteins (MMP-9, N-cadherin, and E-cadherin) expression was verified in coculture model EBL cells after M. bovis infection through WB. Data were relative to coculture model EBL cells without M. bovis infection. (F, G) The impact of coculture model EBL cells after M. bovis infection on migration and invasion capacity was detected using Transwell assay. Data were relative to coculture model EBL cells without M. bovis infection. (H) The detection of EMT-related proteins (MMP-9, N-cadherin, and E-cadherin) of RBMX2 knockout EBL cells after M. bovis-infected BoMac cells via WB. Data were relative to WT EBL cells after M. bovis-infected BoMac cells. (I) The change in the migratory and invasive capabilities of RBMX2 knockout EBL cells after M. bovis-infected BoMac cells was assessed via Transwell assay. Data were relative to WT EBL cells after M. bovis-infected BoMac cells. (J, K) Validate the changes in migration abilities of RBMX2 knockout EBL cells after M. bovis-infected BoMac cells through wound healing assay. Data were relative to WT EBL cells after M. bovis-infected BoMac cells. T-test and two-way ANOVA were used to determine the statistical significance of differences between different groups. Ns presents no significance, * presents p < 0.05, **presents p < 0.01, and *** presents p < 0.001 indicate statistically significant differences. Data were representative of at least three independent experiments.
© 2025, BioRender Inc. Any parts of this image created with BioRender are not made available under the same license as the Reviewed Preprint, and are © 2025, BioRender Inc.
RBMX2 facilitates the EMT in EBL cells via p65/MMP-9 pathway.
(A) Evaluate the impact of pathway activations on expression of EMT-associated proteins (MMP-9, N-cadherin, and E-cadherin) in RBMX2 knockout EBL cells after M. bovis-infected BoMac cells. Data were relative to RBMX2 knockout EBL cells untreated activators. (B, C) Evaluate the impact of pathway activations on the migratory and invasive capabilities of RBMX2 knockout EBL cells after M. bovis-infected BoMac cells via Transwell assay. Data were relative to RBMX2 knockout EBL cells untreated activators. (D, E) Evaluate the impact of pathway activations on the migratory capabilities of RBMX2 knockout EBL cells after M. bovis-infected BoMac cells via wound healing assay. Data were relative to RBMX2 knockout EBL cells untreated activators. (F, G) The impact of p65 silencing on the expression of MMP-9 protein in WT EBL cells after M. bovis infection was assessed using WB. Data were relative to SiRNA-NC in WT EBL cells with M. bovis infection. (H) Predicting the binding ability of RBMX2 promoter and p65 using molecular docking dynamics (I) Verification of RBMX2 promoter region and p65 interaction using dual luciferase reporter system. (I) Using p65 antibody to precipitate p65 protein in EBL cells, and verification of RBMX2 promoter region and p65 interaction using Chip-PCR assay. (K) Predicting potential binding sites for p65 and RBMX2 via protein docking. (L) Verification of MMP-9 promoter region and p65 interaction using dual luciferase reporter system. (M) Verification of MMP-9 promoter region and p65 interaction using Chip-PCR. (N) Predicting potential binding sites for p65 and MMP-9 via protein docking. Two-way ANOVA was used to determine the statistical significance of differences between different groups. *p < 0.05, **p < 0.01, and ***p < 0.001 indicate statistically significant differences. Data were representative of at least three independent experiments.
Protein structure analysis, subfamily localization.
(A) AlphaFold Multimer predicted the sequence and structure of RBMX2 protein. (B) Construction of RBM Gene Family Tree and structural Analysis of genes in RBMX2 subfamily.
The influence of RBMX2 in cell cycle, cell morphology, cell proliferation, and resistance to M. bovis infection.
(A) The different knockout sites of RBMX2 EBL cells were observed by sequencing compared to the bovine RBMX2 sequence or WT EBL cells. (B) Using phalloidine to stain the EBL cell skeleton, observing cell morphology under a high-intensity microscope. The cytoskeleton is labeled with red fluorescence, and the nucleus is stained with blue fluorescence. Scale Bar: 20 μm. (C) Observation of the effect of RBMX2 knockout on cell cycle of EBL cells by flow cytometry assay. Data are represented as the G0/G1 and S phase relative to WT EBL cells. (D) The change in cell number of RBMX2 knockout and silence following 96 and 120 hours of M. bovis infection was observed via crystal violet assay in EBL cells and H1299 cells, respectively. Data were represented as the cell number relative to WT EBL cells and H1299-ShNC cells after M. bovis-infection. Data were representative of at least three independent experiments.
Transcriptome analysis in RBMX2 knockout and WT EBL cells after M. bovis infection in different time points.
(A-C) GO analysis of enriched genes of (A) 0, (B) 24 and (C) 48h after M. bovis infection, respectively. Data were in RBMX2 knockout EBL cells relative to WT EBL cells with M. bovis infection. The changes of these pathways from cell junction-related pathways in 0 hpi to cell proliferation and differentiation-related pathways in 48 hpi. (D-F) KEGG analysis of enriched genes of (D) 0, (E) 24 and (F) 48hpi after M. bovis infection, respectively. Data were in RBMX2 knockout EBL cells relative to WT EBL cells with M. bovis infection. The changes of these pathways from inflammation-related pathways in 0 hpi to cancer-related pathways in 48 hpi.
Transcriptomic analysis of M. bovis infected EBL cells.
(A, B) A volcano map illustrating the transcriptional enrichment genes after WT EBL cells with M. bovis-infection. Data were relative to WT EBL cells without M. bovis infection.
The optimal concentration of activators and their impact on cell viability.
(A-C) Ascertain the optimal concentration of the three pathway activators and their impact on cellular viability via WB and CCK-8 assays. Data were relative to WT EBL cells untreated activators. One-way ANOVA was used to determine the statistical significance of differences between different groups. Ns presents no significance, **presents p < 0.01, and *** presents p < 0.001 indicate statistically significant differences.
RBMX2 enhanced the processes of pathogen adhesion, invasion, and intracellular survival.
(A, B) The impact of M. bovis on the adhesion and invasion of RBMX2 knockout EBL cells following treatment with related-pathway activators, verified by plate counting. Data were relative to RBMX2 knockout EBL cells untreated by activators. (C, D) The impact of M. bovis BCG and M. smegmatis on the adhesion of RBMX2 knockout EBL cells through plate counting assay. Data were relative to WT EBL cells after BCG and M. Smegmatis infection. (E, F) The impact of M. bovis BCG and M. smegmatis on the invasion of RBMX2 knockout EBL cells through plate counting assay. Data were relative to WT EBL cells after BCG and M. Smegmatis infection. (G, H) The impact of Salmonella and E. coli, on the adhesion of RBMX2 knockout EBL cells through plate counting assay. Data were relative to WT EBL cells after Salmonella and E. coli infection. (K, L) The impact of Salmonella and E. coli on the invasion of RBMX2 knockout EBL cells through plate counting assay. Data were relative to WT EBL cells after Salmonella and E. coli infection. One-way and two-way ANOVA were used to determine the statistical significance of differences between different groups. Ns presents no significance, * presents p < 0.05, **presents p < 0.01, and *** presents p < 0.001 indicate statistically significant differences. Data were representative of at least three independent experiments.
RBMX2 facilitates EMT process in EBL cells after M. bovis-infected BoMac cells.
(A) WB detection of changes in mesenchymal cell markers (MMP-9 and N-cadherin) after infection of EBL cells with different infection ratios (10, 20, and 50) of M. bovis. Data were relative to EBL cells without infection of M. bovis. (B) WB detection of the ability of M. bovis infection with macrophages (BoMac cells) to induce epithelial (EBL cells) mesenchymal transition. Data were relative to the addition of PBS in the upper chamber of the coculture model. (C) Staining the skeleton of EBL cells in coculture model after M. bovis infection using ghost pen cyclic peptides. The cytoskeleton is labeled with red fluorescence, and the nucleus is stained with blue fluorescence. Scale Bar: 20 μm. (D) EMT-related mRNAs (MMP-9, N-cadherin, and E-cadherin) expression was verified in coculture model EBL cells after M. bovis infection through RT-qPCR. Data were relative to coculture model EBL cells without M. bovis infection. (E) The detection of EMT-related mRNAs (MMP-9, N-cadherin, and E-cadherin) of RBMX2 knockout EBL cells after M. bovis-infected BoMac cells via RT-qPCR. Data were relative to WT EBL cells after M. bovis-infected BoMac cells. Two-way ANOVA was used to determine the statistical significance of differences between different groups. Ns presents no significance, **presents p < 0.01, and *** presents p < 0.001 indicate statistically significant differences. Data were representative of at least three independent experiments.
RBMX2 facilitates EMT in H1299 cells.
(A) EMT-related proteins expression was verified in H1299 cells after M. bovis infection through WB. Data were relative to H1299 Sh-Con cells. (B) The change in the migratory and invasive capabilities of H1299 cells was assessed via Transwell assay. Data were relative to H1299 Sh-Con cells. Scale Bar: 100 μm. (C) Activation of the MAPK pathway-related protein and p65 protein were activated after RBMX2 knockout and WT EBL cells infected by M. bovis in this coculture model via WB. Data were relative to WT EBL cells with M. bovis infection. Two-way ANOVA was used to determine the statistical significance of differences between different groups. *p < 0.05, **p < 0.01, and ***p < 0.001 indicate statistically significant differences. Data were representative of at least three independent experiments.
