Figure 7.

SLUG and GAS1 cooperated to facilitate NOTCH4 function in maintaining the ML-BCSC characteristics and predicted metastasis and relapse in TNBC patients after chemotherapy. (A) SLUG alone or both SLUG and GAS1 were knocked down in NICD4-overexpressing SUM149 and MDA-MB-231. (B) Apoptosis of the indicated cells in (A) was assessed using flow cytometry. (C) GAS1 alone or both SLUG and GAS1 were overexpressed simultaneously in SUM149 and MDA-MB-231. (D) Apoptosis assay was performed to the indicated cells in (C). Bars showed the percentage of cells distributed in different status, namely, viable cells, early apoptosis (Early-Apop. in short), late apoptosis (Late-Apop. in short) and dead cells. (E) Western blotting to determine the expression of the indicated proteins in sorted NOTCH4^neg and NOTCH4^pos cells of both SUM149 and MDA-MB-231 (neg, negative; pos, positive). (F) Western blotting to determine the expression pattern of the indicated proteins in TNBC and non-TNBC cell lines. β-ACTIN was used as loading control. (G) DMFS and RFS analysis of NOTCH4, SLUG, GAS1 and CCND1 using Kaplan-Meier tool with cohort received chemotherapy. (H) A schematic summary of the findings of this work. NOTCH4 aberrant activation induces and maintains ML-BCSCs status by transcriptionally activating SLUG and GAS1, which work together to endow NOTCH4⁺ cells with enhanced EMT and cellular quiescence, and eventually leading to TNBC metastasis and chemoresistance. *** P<0.001.