Fig. 4

Chronic exposure of TNBC cells to chemotherapy induces WAVE3 expression and activates the CSC phenotype that are associated with chemoresistance. A & B Parental and cisplatin-resistant CIS-R (A) or doxorubicin-resistant DOX-R (B) MDA-MB-231 cells were treated with increased concentrations of cisplatin, ranging from 0 to 100 µM (Cis) and from 0 to 500 nM (Dox) for 2 days followed by MTT assay to assess for cell viability. Data are the mean ± SD (n = 3; **p < 0.01, Student’s t test). C & D Representative Western blot of protein lysates from parental and cisplatin-resistant (C) or doxorubicin-resistant (D) MDA-MB-231 cells that were treated with or without cisplatin (30 µM) or doxorubicin (300 nM) for 2 days and subjected to immunoblotting with antibodies against WAVE3. β-Actin was used as loading control. E & F Quantitative RT-PCR of mRNA of the indicated genes from total RNA isolated from parental and CIS-R (E) or DOX-R (F) MDA-MB-231 cells. Data are the mean ± SD (n = 3, *p < 0.05; **p < 0.01, Student’s t test). G Quantification of volume of tumors derived from inoculation of parental (CT), cisplatin-resistant (CIS-R) or WAVE3-deficient CIS-R (CIS-R-W3KO) MDA-MB-231 cells with treatment with cisplatin (1 mg/kg). The vertical dotted lines indicate the treatment period with cisplatin. Data are generated from five mice per group and each mouse was injected in both the left and right mammary fat pad, resulting in ten tumors. Data are the mean ± SD (n = 10, *p < 0.05; Student’s t test). H Quantification of lung metastatic foci from the animal experiment described in (G). Data are the mean ± SD (n = 5, **p < 0.01; Student’s t test)