Journal: Heliyon

Article Title: Tumor suppressor function of RBMS3 overexpression in EOC associated with immune cell infiltration

doi: 10.1016/j.heliyon.2024.e30603

Figure Lengend Snippet: Primer sequences.

Article Snippet: Prior to being incubated with primary antibodies against RBMS3 (1:2000; No.: DF8599; Affinity Biologicals, China) or GAPDH (1:10,000; No.: AF7021; Affinity Biologicals, China) for an entire night on a shaking table at 4 °C, for 2 h under a condition of the room temperature, 5% skim milk was applied to block the membranes.

Techniques:

Journal: Heliyon

Article Title: Tumor suppressor function of RBMS3 overexpression in EOC associated with immune cell infiltration

doi: 10.1016/j.heliyon.2024.e30603

Figure Lengend Snippet: Antibody information used in the experiment.

Article Snippet: Prior to being incubated with primary antibodies against RBMS3 (1:2000; No.: DF8599; Affinity Biologicals, China) or GAPDH (1:10,000; No.: AF7021; Affinity Biologicals, China) for an entire night on a shaking table at 4 °C, for 2 h under a condition of the room temperature, 5% skim milk was applied to block the membranes.

Techniques:

Journal: Heliyon

Article Title: Tumor suppressor function of RBMS3 overexpression in EOC associated with immune cell infiltration

doi: 10.1016/j.heliyon.2024.e30603

Figure Lengend Snippet: The poor prognosis of OC patients is linked to down-regulated RBMS3 expression in OC tissues. (A) RBMS3 mRNA expression levels from the GEPIA database in both normal tissue and OC. Grey (n = 88) normal samples and red (n = 426) tumor samples. (B, C)Data on OS and PFS for patients with OC were collected with the assistant of the Kaplan-Meier plotter ( https://kmplot.com/analysis/ ). (D) qRT-PCR was used to measure the mRNA levels of RBMS3 in 29 instances of EOC and 23 cases of normal ovarian tissue. (E, F) Western blot analysis reveals the RBMS3 protein expression level in eight pairs of EOC tissues. (G) Typical IHC staining pictures of RBMS3 in normal tissue (Normal) and OC (Tumor). The scale bars are 50 μm and 20 μm respectively. (H, I) Kaplan-Meier study of the effects of 60 EOC patients' RBMS3 expression on OS and PFS. * P < 0.05, ** P < 0.01, *** P < 0.001 and**** P < 0.0001.The uncropped versions of E is in .

Article Snippet: Prior to being incubated with primary antibodies against RBMS3 (1:2000; No.: DF8599; Affinity Biologicals, China) or GAPDH (1:10,000; No.: AF7021; Affinity Biologicals, China) for an entire night on a shaking table at 4 °C, for 2 h under a condition of the room temperature, 5% skim milk was applied to block the membranes.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunohistochemistry

Journal: Heliyon

Article Title: Tumor suppressor function of RBMS3 overexpression in EOC associated with immune cell infiltration

doi: 10.1016/j.heliyon.2024.e30603

Figure Lengend Snippet: Expression of RBMS3 in EOC and normal tissue.

Article Snippet: Prior to being incubated with primary antibodies against RBMS3 (1:2000; No.: DF8599; Affinity Biologicals, China) or GAPDH (1:10,000; No.: AF7021; Affinity Biologicals, China) for an entire night on a shaking table at 4 °C, for 2 h under a condition of the room temperature, 5% skim milk was applied to block the membranes.

Techniques: Expressing

Journal: Heliyon

Article Title: Tumor suppressor function of RBMS3 overexpression in EOC associated with immune cell infiltration

doi: 10.1016/j.heliyon.2024.e30603

Figure Lengend Snippet: The relation between clinical parameters and the expression of RBMS3.

Article Snippet: Prior to being incubated with primary antibodies against RBMS3 (1:2000; No.: DF8599; Affinity Biologicals, China) or GAPDH (1:10,000; No.: AF7021; Affinity Biologicals, China) for an entire night on a shaking table at 4 °C, for 2 h under a condition of the room temperature, 5% skim milk was applied to block the membranes.

Techniques: Expressing

Journal: Heliyon

Article Title: Tumor suppressor function of RBMS3 overexpression in EOC associated with immune cell infiltration

doi: 10.1016/j.heliyon.2024.e30603

Figure Lengend Snippet: The overexpression and suppression of the RBMS3 gene can control the capabilities to migrate, invade, and reproduce of OC cells. (A, B) Stable ID8 cell lines that overexpress and knockdown RBMS3 were created using the lentiviral transfection approach. The fluorescence plot illustrates representative images of stably transfected cells in both dark and bright settings (Microscopic magnification: 20 × ). (C) Comparative mRNA expression levels of RBMS3 in lentiviral-transfected ID8 cells. (D) Western Blot analysis verified that ID8 cells had overexpressed and knocked down RBMS3 genes. (E, F) Plate cloning and CCK-8 were used to determine cell growth. (G, H) Experiments on wound healing demonstrate how RBMS3 affects the capacity for cell migration (Microscopic magnification: 4 × ). (I, J) The Transwell technique for identifying the capacity for the capabilities in invading and migrating of cells (Microscopic magnification: 10 × ). The scale bar is 100 μm * P < 0.05, ** P < 0.01, and *** P < 0.001. The uncropped versions of D is in .

Article Snippet: Prior to being incubated with primary antibodies against RBMS3 (1:2000; No.: DF8599; Affinity Biologicals, China) or GAPDH (1:10,000; No.: AF7021; Affinity Biologicals, China) for an entire night on a shaking table at 4 °C, for 2 h under a condition of the room temperature, 5% skim milk was applied to block the membranes.

Techniques: Over Expression, Control, Knockdown, Transfection, Fluorescence, Stable Transfection, Expressing, Western Blot, Cloning, CCK-8 Assay, Migration

Journal: Heliyon

Article Title: Tumor suppressor function of RBMS3 overexpression in EOC associated with immune cell infiltration

doi: 10.1016/j.heliyon.2024.e30603

Figure Lengend Snippet: The RBMS3 gene induces cell cycle arrest and apoptosis, potentially preventing the progression of OC cells. (A, B) Histograms of the flow cytometry data and flow cytometry detection of the cell cycle distribution of ID8 cells before and after RBMS3 gene overexpression and knockdown. (C, D) Using flow cytometry, the apoptotic rate of ID8 cells with RBMS3 lentivirus overexpression and knockdown was examined. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Prior to being incubated with primary antibodies against RBMS3 (1:2000; No.: DF8599; Affinity Biologicals, China) or GAPDH (1:10,000; No.: AF7021; Affinity Biologicals, China) for an entire night on a shaking table at 4 °C, for 2 h under a condition of the room temperature, 5% skim milk was applied to block the membranes.

Techniques: Flow Cytometry, Over Expression, Knockdown

Journal: Heliyon

Article Title: Tumor suppressor function of RBMS3 overexpression in EOC associated with immune cell infiltration

doi: 10.1016/j.heliyon.2024.e30603

Figure Lengend Snippet: Immune cell infiltration and RBMS3 expression are correlated in OC tissues. (A) TISIDB database data shows the connection between RBMS3 expression and 28 tumor-infiltrating immune cells in human cancer (red indicates positive infiltration, blue indicates negative). (B) The degree of immune cell infiltration in OC tissues is correlated with the expression of RBMS3. (C) An IHC staining diagram showing the relationship between immune cell-related markers and RBMS3 expression (magnification: 400 × ). The scale bar is 20 μm.

Article Snippet: Prior to being incubated with primary antibodies against RBMS3 (1:2000; No.: DF8599; Affinity Biologicals, China) or GAPDH (1:10,000; No.: AF7021; Affinity Biologicals, China) for an entire night on a shaking table at 4 °C, for 2 h under a condition of the room temperature, 5% skim milk was applied to block the membranes.

Techniques: Expressing, Immunohistochemistry

Journal: Heliyon

Article Title: Tumor suppressor function of RBMS3 overexpression in EOC associated with immune cell infiltration

doi: 10.1016/j.heliyon.2024.e30603

Figure Lengend Snippet: The relationship between high and low RBMS3 expression in ID8 cells and immune cell markers. (A,B) RBMS3 expression in ID8 cells in both groups. (C,D) Immunofluorescence imagesof INOS, ARG1, FOXP3 and CD11b in cells of Lv-Vector and Lv-RBMS3 groups (200 × and 400 × ). The scale bars are 100 μm and 50 μm respectively.

Article Snippet: Prior to being incubated with primary antibodies against RBMS3 (1:2000; No.: DF8599; Affinity Biologicals, China) or GAPDH (1:10,000; No.: AF7021; Affinity Biologicals, China) for an entire night on a shaking table at 4 °C, for 2 h under a condition of the room temperature, 5% skim milk was applied to block the membranes.

Techniques: Expressing, Immunofluorescence, Plasmid Preparation

Journal: Heliyon

Article Title: Tumor suppressor function of RBMS3 overexpression in EOC associated with immune cell infiltration

doi: 10.1016/j.heliyon.2024.e30603

Figure Lengend Snippet: Impact of overexpressing the RBMS3 gene on in vivo angiogenesis and tumor growth. (A) Diagram of mouse subcutaneous tumor growth. (B) Tumor weight 28 days after inoculating cells. (C) Mouse weight growth curve. (D) Measure the subcutaneous tumor volume, tumor volume = 0.5 × largest dimension × shortest dimension 2 . (E, F) Immunohistochemistry (IHC) staining of Ki-67 and CD31 in transplanted tumor tissue Lv-Vector and Lv-RBMS3 groups (200 × and 400 × ). The scale bars are 50 μm and 20 μm respectively.

Article Snippet: Prior to being incubated with primary antibodies against RBMS3 (1:2000; No.: DF8599; Affinity Biologicals, China) or GAPDH (1:10,000; No.: AF7021; Affinity Biologicals, China) for an entire night on a shaking table at 4 °C, for 2 h under a condition of the room temperature, 5% skim milk was applied to block the membranes.

Techniques: In Vivo, Immunohistochemistry, Plasmid Preparation

Journal: Heliyon

Article Title: Tumor suppressor function of RBMS3 overexpression in EOC associated with immune cell infiltration

doi: 10.1016/j.heliyon.2024.e30603

Figure Lengend Snippet: The relationship between high and low expression of RBMS3 in tumor tissues and immune cell markers. (A,B) IHC staining detects the expression of RBMS3 in mouse tumor tissues. (C,D) Immunofluorescence detection images of INOS, ARG1, FOXP3 and CD11b in the Lv-Vector and Lv-RBMS3 groups of transplanted tumor tissues (200 × and 400 × ). The scale bars are 50 μm and 20 μm respectively.

Article Snippet: Prior to being incubated with primary antibodies against RBMS3 (1:2000; No.: DF8599; Affinity Biologicals, China) or GAPDH (1:10,000; No.: AF7021; Affinity Biologicals, China) for an entire night on a shaking table at 4 °C, for 2 h under a condition of the room temperature, 5% skim milk was applied to block the membranes.

Techniques: Expressing, Immunohistochemistry, Immunofluorescence, Plasmid Preparation

Journal: Heliyon

Article Title: Tumor suppressor function of RBMS3 overexpression in EOC associated with immune cell infiltration

doi: 10.1016/j.heliyon.2024.e30603

Figure Lengend Snippet: Effects of RBMS3 on the Wnt/β-catenin signalling pathway in ovarian cancer cells. (A, B) Up-regulation of RBMS3 regulates the Wnt/β-catenin signalling pathway in ID8 cells. (C) After overexpression of RBMS3 and activation of the Wnt/β-catenin signalling pathway, the migratory activity of ID8 cells was detected by transwell. (D) After overexpression of RBMS3 and activation of the Wnt/β-catenin signalling pathway, the invasive activity of ID8 cells was detected by transwell. The scale bars is 100 μm ** P < 0.01, *** P < 0.001 and **** P < 0.0001. The uncropped versions of A is in .

Article Snippet: Prior to being incubated with primary antibodies against RBMS3 (1:2000; No.: DF8599; Affinity Biologicals, China) or GAPDH (1:10,000; No.: AF7021; Affinity Biologicals, China) for an entire night on a shaking table at 4 °C, for 2 h under a condition of the room temperature, 5% skim milk was applied to block the membranes.

Techniques: Over Expression, Activation Assay, Activity Assay

Journal: Cancer Medicine

Article Title: The RNA ‐binding protein RBMS3 inhibits the progression of colon cancer by regulating the stability of LIMS1 mRNA

doi: 10.1002/cam4.7129

Figure Lengend Snippet: RBMS3 is down‐regulated in colon cancer tissues. (A) Cluster analysis of RBM family genes using data from TCGA and GTEx databases reveals three clusters. Genes in Cluster 1 are significantly downregulated in tumors, genes in Cluster 2 show no significant difference in expression between colon cancer and normal colon tissues, and genes in Cluster 3 are highly expressed in colon cancer tissues. (B) The expression levels of 8 RBM family genes in normal colon tissues and colon cancer tissues were analyzed using t ‐tests. The results show a significant difference in expression between the two groups *** p < 0.001. (C) The correlation between RBMS1 and RBMS3 expression and clinical stage in colon cancer was analyzed using analysis of variance (ANOVA). A p ‐value <0.05 indicates a significant correlation. (D) The expression levels of RBMS1 and RBMS3 in colon cancer tissues and normal colon tissues were detected using Western blot analysis. Representative images are shown here. A total of 20 pairs of tissues were tested. (E) The grayscale values of Western blot strips from 20 paired colon cancer and normal colon tissues were statistically analyzed using t ‐tests, * p < 0.05, *** p < 0.001. (F) qRT‐PCR analysis was performed to evaluate the expression of RBMS1 and RBMS3 in colon cancer tissues and normal colon tissues, *** p < 0.001. (G, H) IHC analysis was conducted to examine the expression of RBMS3 in colon cancer tissues and normal colon tissues. Statistical analysis was conducted using t ‐tests to assess the IHC scores. *** p < 0.001.

Article Snippet: We conducted an IHC assay to discern protein expression in human paraffin‐embedded colon cancer samples using anti‐RBMS3 (1:100, Abcam, Cambridge, UK, No. ab272612).

Techniques: Expressing, Western Blot, Quantitative RT-PCR

Journal: Cancer Medicine

Article Title: The RNA ‐binding protein RBMS3 inhibits the progression of colon cancer by regulating the stability of LIMS1 mRNA

doi: 10.1002/cam4.7129

Figure Lengend Snippet: Overexpression of RBMS3 inhibits colon cancer proliferation. (A) Western blot analysis was conducted to evaluate the expression level of RBMS3 in both PLVX and RBMS3 overexpression cell models, PLVX was used as the control group. (B) The grayscale intensities of the Western blot bands were statistically analyzed, * p < 0.05. (C) RBMS3 expression was examined through qRT‐PCR in the PLVX control group and the group with RBMS3 overexpression, *** p < 0.001. (D) Cell proliferation was assessed using the MTT assay in both the RBMS3 overexpression group and the PLVX control group, ** p < 0.01. (E) Cell proliferation was analyzed using the EDU assay in the RBMS3 overexpression group and the PLVX control group. (F) An experimental study employing a subcutaneous murine model was conducted to assess the growth condition of HCT116 cells inoculated with overexpressed RBMS3, knocked‐down RBMS3, and the control. Six mice were used in each group for the analysis. (G) Tumor weight was compared among the control group, RBMS3 group, and RBMS3‐sh1 group, * p < 0.05, ** p < 0.01. (H) The tumor growth curve over time was plotted for the control, RBMS3, and RBMS3‐sh1 groups, * p < 0.05, ** p < 0.01.

Article Snippet: We conducted an IHC assay to discern protein expression in human paraffin‐embedded colon cancer samples using anti‐RBMS3 (1:100, Abcam, Cambridge, UK, No. ab272612).

Techniques: Over Expression, Western Blot, Expressing, Quantitative RT-PCR, MTT Assay, EdU Assay

Journal: Cancer Medicine

Article Title: The RNA ‐binding protein RBMS3 inhibits the progression of colon cancer by regulating the stability of LIMS1 mRNA

doi: 10.1002/cam4.7129

Figure Lengend Snippet: RBMS3 suppresses migration and invasion of colon cancer cells. (A, B) The transwell migration and invasion assays revealed that the overexpression of RBMS3 exerts an inhibitory effect on the migration and invasion of HCT15 and HCT116 cells (magnification, 100×; scale bars, 200 μm). (C, D) Statistical analysis of the cell migration and invasion among different groups was conducted using the t ‐test, *** p < 0.001, **** p < 0.0001. (E, F) The scratch assay was employed to assess the migration of HCT15 cells in the overexpressed RBMS3, knocked‐down RBMS3, and control groups. (G, H) Statistical analysis was performed to evaluate the migration area of HCT15 cells in the overexpressed RBMS3, knocked‐down RBMS3, and control groups at different time points, * p < 0.05, ** p < 0.01. (I) The number of lung metastatic lesions observed under the microscope in the overexpressed RBMS3, knocked‐down RBMS3, and control groups in the tail vein lung metastasis model were statistically analyzed using the t ‐test, * p < 0.05. (J) The tail vein injection model was used to examine the influence of RBMS3 on the lung colonization of HCT116 cells in vivo, with a total of ten mice in each group. (magnification, 20×; scale bars, 500 μm; the black arrow represents the location of the metastasis).

Article Snippet: We conducted an IHC assay to discern protein expression in human paraffin‐embedded colon cancer samples using anti‐RBMS3 (1:100, Abcam, Cambridge, UK, No. ab272612).

Techniques: Migration, Over Expression, Wound Healing Assay, Microscopy, Injection, In Vivo

Journal: Cancer Medicine

Article Title: The RNA ‐binding protein RBMS3 inhibits the progression of colon cancer by regulating the stability of LIMS1 mRNA

doi: 10.1002/cam4.7129

Figure Lengend Snippet: LIMS1 is the key target gene regulated by RBMS3. (A) Differential expression analysis was performed on the RNA‐seq data comparing the overexpression RBMS3 HCT15 cells and the control cells (pLVX group), as well as the shRBMS3 HCT15 cells and the control cells (PLKO group). The overexpression RBMS3 group showed 2576 upregulated genes and 3796 downregulated genes, while the shRBMS3 group exhibited 872 downregulated genes and 184 upregulated genes (cutoff: FC > 1.5 or FC < 0.75, p adj < 0.05). (B) In the 506 genes that were upregulated in the overexpression of RBMS3 and downregulated in the knockdown of RBMS3, and in the 103 genes that were downregulated in the overexpression of RBMS3 and upregulated in the knockdown of RBMS3, intersection analysis was performed with the predicted RBMS3 binding genes in the RNA‐INTER database, resulting in 432 candidate genes. (C) A GO enrichment analysis of the 432 candidate genes was conducted using the Metascape database. (D) A RIP assay was performed to measure the enrichment fold of RBMS3 binding target genes using HCT15 cells, * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant. (E) Dual luciferase assays confirmed the regulatory effect of RBMS3 on LIMS1 using HCT15 cells, * p < 0.05. (F) Western blot analysis was performed to assess the expression of LIMS1 in the control group and RBMS3 HCT15 cells, demonstrating increased LIMS1 expression with RBMS3 overexpression and decreased expression with shRBMS3 HCT15 cells (knockdown of RBMS3). (G) Treatment with Actinomycin D for 12 h revealed enhanced mRNA stability of LIMS1 in the RBMS3 overexpression group compared to the control group of HCT116 cells. (H) The stability of LIMS1 mRNA in the HCT116 cell line decreased upon RBMS3 knockdown, as observed after a 12‐h treatment with actinomycin D.

Article Snippet: We conducted an IHC assay to discern protein expression in human paraffin‐embedded colon cancer samples using anti‐RBMS3 (1:100, Abcam, Cambridge, UK, No. ab272612).

Techniques: Expressing, RNA Sequencing Assay, Over Expression, Binding Assay, Luciferase, Western Blot

Journal: Cancer Medicine

Article Title: The RNA ‐binding protein RBMS3 inhibits the progression of colon cancer by regulating the stability of LIMS1 mRNA

doi: 10.1002/cam4.7129

Figure Lengend Snippet: Downregulated LIMS1 recovers the effect of RBMS3 overexpression in colon cancer. (A) Western blot analysis was conducted to evaluate the expression level of LIMS1 in PLVX, RBMS3, and RBMS3+shLIMS1 cell models. (B) The grayscale intensities of the Western blot bands were statistically analyzed, ** p < 0.01, *** p < 0.001. (C) Rescued effects of LIMS1 on cell proliferation of HCT15 and HCT116 cells upon RBMS3 overexpression were evaluated. All cells were harvested for MTT analyses at the indicated time points. Data are presented as mean ± SD, *** p < 0.001. (D, E) Rescued effects of LIMS1 on migration and invasion of HCT15 and HCT116 cells upon RBMS3 overexpression were evaluated (magnification, 100×; scale bars, 200 μm). Data are presented as means ± SD, ** p < 0.01, *** p < 0.001.

Article Snippet: We conducted an IHC assay to discern protein expression in human paraffin‐embedded colon cancer samples using anti‐RBMS3 (1:100, Abcam, Cambridge, UK, No. ab272612).

Techniques: Over Expression, Western Blot, Expressing, Migration