Fig. 3: RNF40 is indispensable for the stemness and drug-tolerant features of BLBC cells.

A Dot plot of the RNF40 gene essentiality in various human BLBC cell lines, as assessed by the Crispr library screening “Avana” from DepMap (source: https://depmap.org/portal/). B, C Real-time quantitative PCR (RT-qPCR) and western blot analysis of RNF40 and H2Bub1 in siControl- and siRNF40-treated HCC1806 cells at 48 h of RNF40 silencing. D Volcano plot displaying gene expression changes occurring in HCC1806 cells upon RNF40 depletion (48 h of RNF40 silencing) and measured by mRNA sequencing (down: log2FC ≤ −0.7, p < 0.05, up: log2FC ≥ 0.7, p < 0.05, basemean≥15). E GSEA profile of the “RAMALHO_STEMNESS_UP” gene signature, significantly impaired in siRNF40-treated HCC1806 cells (source: https://www.gsea-msigdb.org/gsea/msigdb/). NES normalized enrichment score, FDR false discovery Rate. Proliferation kinetics (F, G), colony (H, I) and tumorsphere formation assay (J, K) in siControl- and siRNF40-treated HCC1806 and HCC1937 cells. L CAM assay of siControl- and siRNF40-treated HCC1806 cells. The micro-CT scans of the tumors (left panel) and respective quantification of their volumes (right panel) are provided for both conditions. M Dose response assay with increasing concentration of cisplatin in siControl- or siRNF40-treated HCC1806 cells. Representative stained cells (left panel) and dose response curves with the respective IC50 of cisplatin per condition (right panel) is provided. N Receiver Operating Characteristic (ROC) plot showing the poor response of RNF40^high-expressing TNBC patients to any chemotherapy (expression array id: 206845_s_at, source: http://www.rocplot.org/). Statistical test: Student t-test (B, M for the AUC, F–M for the number of colonies, spheres or tumors), Mann–Whitney test (H–K for colony or sphere size).^*p < 0.05, ^**p < 0.01, ^***p < 0.005. Error bars: Standard error of the mean (SEM). All experiments were performed in biological triplicates per condition.