Fig 2: In vivo anti-tumor activity of Notch1-saRNA-1480. (A) Representative images of nude mice. (B) Representative images of tumors. (C) Tumor growth curves. (D) The internal structure of the tumor by hematoxylin and eosin staining. (E) Western blot results and analysis showing the expression levels of (F) Notch1, (G) VEGFR2, (H) AR and (I) VEGF in tumor tissues. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. sa, small activating; VEGFR, vascular endothelial growth factor receptor; AR, androgen receptor

Fig 3: Notch1-Jagged signaling regulates macrophage contact-induced stemness in breast cancer cells.a Quantification of SORE6+ cells in TC + Mac co-cultures treated with DMSO or DAPT. n = 60 fields at ×20 magnification; data normalized to the DAPT mean; unpaired two-tailed Student’s t-test, **p = 0.0061. b Quantification of SORE6+ cells in TC + Mac co-cultures treated with DMSO or 20 µM SAHM1. n = 48 (DMSO), 47 (SAHM1) fields at ×20 magnification; data normalized to the SAHM1 mean; two-sided Mann–Whitney test, **p = 0.0025. c Western blotting showing Notch1 KD in Notch1 siRNA-treated SORE6 MDA-MB-231 cells after 48 h. It is noteworthy that Notch2 levels are unaffected by Notch1 siRNA. Source data are provided as a Source Data file. d Quantification of SORE6+ cells in TC + Mac co-culture assay. Tumor cells were treated with either control siRNA or Notch1 siRNA for 36 h and co-cultured with macrophages overnight. Measurements were made 48 h post siRNA transfection. n = 32 fields of 330 × 330 µm2; data normalized to the Notch1si condition mean; unpaired two-tailed Student’s t-test, *p = 0.0232. e Quantification of SORE6+ cells in tumor cells (MDA-MB-231-expressing tdTomato and with SORE6>GFP) treated with either Jagged1 scramble peptide or functional Jagged1 peptide. n = 80 fields at ×20 magnification; data normalized to the TC + Jag1 scramble mean; unpaired two-tailed Student’s t-test, **p = 0.0012. f Change in % SORE6+ cells after co-culture with either wild-type BACs compared to tumor cells alone (40% increase, data from Fig. 4d, unshaded box) or Jag1KO BACs compared to wild-type BACs (no change, shaded box). n = 50 (wild-type BACs) and 53 (Jag1KO BAC) fields at ×20 magnification; two-sided Mann–Whitney test, *p = 0.033. g Change in % SORE6+ cells after co-culture with either wild-type BACs compared to tumor cells alone (40% increase, data from Fig. 4d, unshaded boxes) or Jag1/Jag2 antibody treatments compared to IgG-treated sample (no change, shaded boxes). n = 54 (IgG), 57 (Jag1), 56 (Jag2), and 55 (Jag1 + Jag2) fields at ×20 magnification; two-sided Mann–Whitney test, *p-values: Jag1 = 0.035, Jag2 = 0.013, Jag1 + Jag2 = 0.02. h Quantification of changes in Sox9Hi cells in fixed PyMT mammary tumor after PBS (control) or DAPT treatment. n = 5 and 7 mice for control and DAPT conditions; two 2–6 mm2 tumor tissue areas were analyzed in each mouse; unpaired two-tailed Student’s t-test, ****p < 0.0001. i Quantification of changes in macrophage density in fixed PyMT mammary tumor after PBS (control) or DAPT treatment. n = 5 and 7 mice for control and DAPT conditions; two 2–6 mm2 tumor tissue areas were analyzed in each mouse; unpaired two-tailed Student’s t-test, n.s. p = 0.41. In all quantifications, boxes indicate 25th–75th percentile interquartile range (IQR) and central line indicates the median. Top and bottom whiskers were plotted using Tukey’s method and extend to 75th percentile + 1.5 × IQR and 25th percentile - 1.5 × IQR, respectively. Points below and above the whiskers are drawn as individual dots.

Fig 4: Overexpression of miR-144-3p aggravates hypoxia-induced injury by regulating Notch1 in the H9c2 cells. (A) The expression of Notch1 protein was measured in the H9c2 cells transfected with agomiR-NC, agomiR-144-3p, agomiR-144-3p and pc-Notch1 or pc-NC or NC group after hypoxia treatment for 24 h by the Western blot analysis. Cell viability (B), apoptosis (C) and apoptotic protein levels (D) were detected in the H9c2 cells transfected with agomiR-NC, agomiR-144-3p, agomiR-144-3p and pc-Notch1 or pc-NC or NC group after hypoxia treatment for 24 h by the trypan blue exclusion method, flow cytometry or Western blot assay, respectively. *P < 0.05 compared with the control, agomiR-NC or agomiR-144-3p and pc-NC group.

Fig 5: miR-34a directly targets Notch1. (A) Interaction between miR-34a and 3'UTR of Notch1 was predicted by TargetScan. (B) Luciferase activity of a reporter containing a Notch1 3'UTR-WT or 3'UTR-MUT (with a mutation in the miR-34a binding site). (C) Western blots and (D) quantified expression of Notch1 protein in MCF-7 cells in different groups, expressed as the fold of the NC group. (E) Relative mRNA expression level of Notch1 in MCF-7 cells in different groups. *P<0.05 and **P<0.01 vs. NC group. miR, microRNA; NC, negative control; UTR, untranslated region; WT, wild-type; MUT, mutated.