Figure 1.

Multi-drug-resistant and breast cancer stem cell-like features of the newly established 4T1/epirubicin (EPB) cells and expanded ubiquitinated protein (UP) spectrum with related markers and therapeutic target-associated proteins. (A) The cell viability of 4T1/WT cells and 4T1/EPB cells was assessed at varying concentrations of EPB, cisplatin, Taxol, and 5-fluorouracil, respectively, to calculate IC50 values and resistance index. (B) Scheme of the in vivo drug sensitivity experimental protocol. (C) Tumor growth and tumor weight of tumor-bearing mice (n = 6 per group) in (B). (D) qRT-PCR analysis was applied to the expression of MDR1, BCRP, GST-π, and MMP7 in 4T1/WT and 4T1/EPB cells. (E) The expression of CD44^highCD24^low population and ALDH1 activity on 4T1/WT cells and 4T1/EPB cells was determined by flow cytometry. (F) The tumorigenicity of 4T1/WT cells and 4T1/EPB cells in BALB/c mice was detected (n = 5 per group). (G) The equal protein loading of the protein marker (lane 0), whole cell lysate (lane 1 and lane 4), unbound lysate (lane 2 and lane 5), and α-Al₂O₃-Vx3-UPs (lane 3 and lane 6) from 4T1/WT cells and 4T1/EPB cells was verified by SDS-PAGE electrophoresis and Coomassie blue stain. (H) The protein levels of the ubiquitin in (G) were determined by western blot. (I) Venn diagram showing the overlap between the 362 proteins identified in UPs from 4T1/WT cells by LC–MS/MS analysis (green) and the 2,125 proteins identified in UPs from 4T1/EPB cells (red). (J) Bar chart showing the number of proteins classified according to the following categories: drug resistance markers, cancer stem cell markers and therapeutic targets, and other proteins in (I). P-values were determined by Mann–Whitney U-test. The results are representative of three independent experiments, and data were expressed as means ± SEM (**p < 0.01; ns, not significant).