Fig 1: RAD18 promotes TNBC cell proliferation and maintains the stemness of TNBC in vitro.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.
Fig 2: RAD18High TNBC promotes M2-like TAM polarization via TGF-ß secretion and JNK pathway inhibition, inducing more TGF-ß secreted from M2-like TAM reciprocally upregulates RAD18 to form a positive regulatory loop in the TNBC-TAM interaction.A, B qRT-PCR was used to detect the expression of cytokines in both TNBC cells and macrophages after co-cultivation. The THP1- derived Mf co-cultured with shNC and shRAD18 MDA-MB-231 for 3 d. The primary THP-1-derived Mf (M0) was used as control (B). C Gene set enrichment analysis plot showing enriched TGF-ß signaling pathway in the TCGA cohorts. D Detection of TGF-ß in co-cultured supernatant by ELISA. E WB detection of protein expression levels of CD163, PPAR-d, NF-?Bp65, NF-?Bp-p65, JNK, p-JNK, and c-Jun in macrophages. F ELISAs were used to determine the TGF-ß concentration in supernatant secreted by macrophage alone after co-cultivation. G, H The tumor sphere formation and protein levels of stemness-related signature genes (CD44, OCT4, SOX2, Nanog) were detected to verify the stemness of shNC/shRAD18 MDA-MB-231 co-cultured with or without macrophages in the absence or presence of a neutralizing antibody specific for TGF-ß are presented. *p < 0.05, **p < 0.01, ***p < 0.001. All experiments were performed independently at least three times.
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