Activation of Notch signaling in both senescent and adjacent non-senescent breast cancer MCF-7 cells. (A) Scheme showing the experimental setup. Flow cytometry sorting of collected breast cancer cells after co-culture or monoculture. (B) Immunoblot analysis of protein expression of active Notch1 intracellular domain (N1ICD) and the canonical Notch target, HES1, assessed in MCF7-mRFP cells subjected to the indicated treatments. (C) RT-qPCR analysis of expression of target genes HES1 and HEY1 and JAG1 in MCF7-mRFP cells subjected to the indicated treatments (error bars indicate mean SD, n = 3 experimental replicates,* p < 0.05, ** p < 0.01). (D) Immunoblot analysis of protein expression of active Notch1 intracellular domain (N1ICD) and the canonical Notch1target, HES1, assessed in MCF-7 cells subjected to the indicated treatments. (E) RT-qPCR analysis of gene expression of target genes HES1 and HEY1 and the JAG1 ligand of Notch in MCF-7 cells subjected to the indicated treatments (error bars indicate the mean SD, n = 3 experimental replicates,* p < 0.05, ** p < 0.01).