Fig. 2

Treatment with AD-01 or ALM201 downregulates DLL4 and/or Notch 4 expression. The anti-CSC effect of ALM201 appears independent of CD44. Western blot of cell lysate from MDA-MB-231 cells treated with AD-01 (100 nM) and probed for DLL4 (a), Notch4ICD (b), and GAPDH (representative blot in inset; Notch4ICD as double band). Relative protein expression was quantified using ImageJ (t-test). c Real-time qPCR analysis of DLL4 mRNA levels in MDA-MB-231 cells treated with ALM201 (100 nM). The difference in gene expression was presented as a fold change relative to the expression of the housekeeping genes, GAPDH and ß –Actin (t-test). d Mammosphere formation in stable CD44 knockdown (KD) MDA-MB-231 cells treated with ALM201 (100 nM) vs. parental (MDA-MB-231) cells (one-way ANOVA with post-hoc Dunnett’s multiple comparisons test). Real-time qPCR analysis of CD44 mRNA levels in stable CD44 KD MDA-MB-231 cells vs. parental control; n = 1. Data points are mean ± SEM. n ≥ 3. * p < 0.05, ** p < 0.01, ***p < 0.01. ICD – intracellular domain