Fig 2: The expression and prognostic associations of RGS10 in breast cancer.(A) RGS10 mRNA levels in 31 normal human tissues. Data were derived from the Genotype-Tissue Expression database. (B) RGS10 mRNA levels in cell lines representing 21 human cancers. Data were derived from the Cancer Cell Line Encyclopedia database. (C) qRT-PCR showing RGS10 mRNA levels in freshly resected breast cancer tissues (n = 20) and matched adjacent normal breast tissues. **p<0.01, Student’s t -test. (D, E) Survival analyses showing disease-free survival (DFS) (D) and overall survival (OS) (E) in patients with breast cancer stratified by high versus low RGS10 mRNA levels. Data were derived from the Kaplan–Meier plotter database. (F) Representative images showing immunohistochemical staining of RGS10 protein expression in breast cancer tissues or normal tissues (n = 133) (magnification: ×200 and ×400). (G, H) Kaplan–Meier analysis showing DFS (G) and OS (H) in patients with breast cancer stratified by presence versus absence of RGS10 protein in breast cancer tissues (n = 133). Figure 1—source data 1.Original files for Figure 1.

Fig 3: Protein–protein network interaction network and Gene Ontology (GO) functional- and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway-enrichment analysis of genes co-expressed with RGS10.(A) Volcano plot showing differentially expressed genes between SKBR3 cells transfected with shRNA-RGS10 or shRNA-NC. (B–E) KEGG pathway analysis and GO enrichment analysis of differentially expressed genes showing the ten most enriched terms. BP: biological processes; MF: molecular function; CC: cellular compartment. (F) LCN2, E-cadherin, and vimentin protein levels in SKBR3 cells transfected with shRNA-RGS10 or shRNA-NC. The original files of the full raw unedited blots are provided in Figure 3—source data 1. The uncropped gels with the relevant bands labeled are provided in Figure 3—source data 2. The statistical data is provided in Figure 3—source data 3. (G) Schematics of predicted MIR539-5p binding sites between wild-type and mutant RGS10 sequences in the 3ʹ-untranslated regions. (H) Relative luciferase activities detected after cotransfection of wild-type or mutant luciferase reporter plasmids and an MIR539-mimic. Figure 3—source data 1.Original files for the gels in Figure 3F. Figure 3—source data 2.Uncropped gels with the relevant bands labeled in Figure 3F. Figure 3—source data 3.Statistical data for Figure 3F. Figure 3—source data 4.Original files for Figure 3A–E, G and H.

Fig 4: RGS10 silencing increases the proliferation and migration of breast cancer cells in vitro.(A) Western blotting showing RGS10 protein levels in molecularly distinct breast cancer cell lines. The original files of the full raw unedited blots are provided in Figure 2—source data 1. The uncropped gels with the relevant bands labeled are provided in Figure 2—source data 2. The statistical data is provided in Figure 2—source data 3. (B) Western blotting showing RGS10 protein levels in SKBR3 cells transfected with three independent shRNA constructs, shRNA-RGS10-161, shRNA-RGS10-321, and shRNA-RGS10-506, and shRNA-NC. The original files of the full raw unedited blots are provided in Figure 2—source data 4. The uncropped gels with the relevant bands labeled are provided in Figure 2—source data 4. The statistical data is provided in Figure 2—source data 6. (C) CCK-8 assay showing the viability of SKBR3 cells transfected with shRNA-RGS10-161, shRNA-RGS10-506, or shRNA-NC. **p <0.01, one-way ANOVA. (D–F) Colony formation (D) and transwell migration/invasion (E, F) assays in SKBR3 cells transfected with shRNA-RGS10-161, shRNA-RGS10-506, or shRNA-NC. ***p<0.001, one-way ANOVA. Figure 2—source data 1.Original files for the gels in Figure 2A. Figure 2—source data 2.Uncropped gels with the relevant bands labeled in Figure 2A. Figure 2—source data 3.Statistical data for Figure 2A. Figure 2—source data 4.Original files for the gels in Figure 2B. Figure 2—source data 5.Uncropped gels with the relevant bands labeled in Figure 2B. Figure 2—source data 6.Statistical data for Figure 2B. Figure 2—source data 7.Original files for Figure 2C. Figure 2—source data 8.Original files for Figure 2D. Figure 2—source data 9.Original files for Figure 2E. Figure 2—source data 10.Original files for Figure 2F.

Fig 5: MIR539-5p regulates the migration, invasion, proliferation, and epithelial–mesenchymal transition (EMT) of breast cancer cells.(A) qPCR showing the transfection efficiency of the MIR539-5p mimic. ***p<0.001, one-way ANOVA. (B, C) qRT-PCR and western blotting showing RGS10 mRNA and protein levels in SKBR3 cells transfected with the MIR539-5p mimic, negative control (NC), or wild type (WT). ***p<0.001, one-way ANOVA. The original files of the full raw unedited blots are provided in Figure 4—source data 1. The uncropped gels with the relevant bands labeled are provided in Figure 4—source data 2. The statistical data is provided in Figure 4—source data 3. (D) CCK-8 assay showing the viability of SKBR3 cells transfected with the MIR539-5p mimic, NC, or WT. ***p<0.001, one-way ANOVA. (E–G) Colony formation (E) and transwell migration/invasion (F, G) assays in SKBR3 cells transfected with the MIR539-5p mimic, NC, or WT. *p<0.05, **p<0.01, Student’s t-test. (H) Western blotting showing protein levels of LCN2 and biomarkers of EMT in SKBR3 cells transfected with the MIR539-5p mimic or NC. The original files of the full raw unedited blots are provided in Figure 4—source data 4. The uncropped gels with the relevant bands labeled are provided in Figure 4—source data 5. The statistical data is provided in Figure 4—source data 6. (I) Immunofluorescence staining showing E-cadherin, vimentin, and snail protein expression in SKBR3 cells transfected with the MIR539-5p mimic or NC. Scale bar: 50 µm. Figure 4—source data 1.Original files for the gels in Figure 4C. Figure 4—source data 2.Uncropped gels with the relevant bands labeled in Figure 4C. Figure 4—source data 3.Statistical data for Figure 4C. Figure 4—source data 4.Original files for the gels in Figure 4H. Figure 4—source data 5.Uncropped gels with the relevant bands labeled in Figure 4H. Figure 4—source data 6.Statistical data for Figure 4H. Figure 4—source data 7.Original files for Figure 4A, B and D. Figure 4—source data 8.Original files for Figure 4E. Figure 4—source data 9.Original files for Figure 4F. Figure 4—source data 10.Original files for Figure 4G. Figure 4—source data 11.Original files for Figure 4I.