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. 2022 Apr 12;8:196. doi: 10.1038/s41420-022-00968-9

Fig. 2. RAD18 promotes TNBC cell proliferation and maintains the stemness of TNBC in vitro.

Fig. 2

RAD18 increases TNBC cell proliferation and maintains the CSC stemness in vitro. A RAD18 protein levels in TNBC cells transfected with RAD18 shRNA lentivirus were determined by western blotting. B Cells were seeded in a 96-well plate and CCK-8 assay was used to detect cell proliferation at indicated hours (24, 48, 72, 96, and 120 h). C colony formation assay was performed to examine cell proliferation. Cells were seeded in six-well plates at a density of 2000 cells per well and cultivated for 2w. D An EdU assay was performed to determine the proliferation of different groups of TNBC cells. E Cell apoptosis in shNC and shRAD18 TNBC cells were assessed by Annexin V/7‐AAD flow cytometric analysis. F Representative flow cytometric analysis of CD44+/CD24− BCSC population percentages in shNC and shRAD18 TNBC cells. G The tumor sphere formation in TNBC cells treated as indicated (200×). The quantification of mammospheres were counted using a microscope with size ≥50 µm. H, I mRNA and protein levels of stemness-related signature genes (CD44, OCT4, SOX2, Nanog). GAPDH was analyzed as a loading control. *p < 0.05, **p < 0.01, ***p < 0.001. All experiments were performed independently at least three times.