NOTCH1 combined with chemotherapy synergistically inhibits triple-negative breast cancer

Figure 1 Knockdown of NOTCH1 inhibits the proliferation and migration of TNBC cells in vitro. A: After viral transduction of MDA-MB-231 cells, successfully transduced cells exhibited green fluorescence under fluorescence microscopy; B and C: RT-PCR and western blotting validation of the knockdown efficiency of NOTCH1 in stable MDA-MB-231 cells; D: CCK-8 assay results show that the proliferation of stable NOTCH1-knockdown MDA-MB-231 cells is reduced compared to wild-type MDA-MB-231 cells; E: Colony formation assay showing that colony formation of stable NOTCH1-knockdown MDA-MB-231 cells was reduced compared to wild-type MDA-MB-231 cells; F: Transwell assay showing that the invasion of stable NOTCH1-knockdown MDA-MB-231 cells was reduced compared to wild-type MDA-MB-231 cells; G: Immunofluorescence showing that after NOTCH1 knockdown, the filamentous arrangement of F-actin in MDA-MB-231 cells was suppressed, and the lamellipodia-like protrusions around the cell periphery were reduced. ^aP < 0.001.

Figure 2 Knockdown of NOTCH1 enhances the chemosensitivity of triple-negative breast cancer cells to cisplatin. A: Analysis of cell sensitivity to cisplatin showing that, compared to control cells (IC₅₀ = 15.36 μM), stable NOTCH1-knockdown MDA-MB-231 cells exhibited higher sensitivity to cisplatin treatment (IC₅₀ = 10.20 μM); B: CCK-8 assay indicating that the combination of NOTCH1 knockdown and cisplatin treatment significantly inhibited cell proliferation; C: TUNEL assay showing that after NOTCH1 knockdown and cisplatin treatment, the proportion of apoptotic cells significantly increases. ^aP < 0.05; ^bP < 0.01.

Figure 3 NOTCH1 and NT5E are positively correlated in triple-negative breast cancer cells. A and B: Western blot and RT-PCR results show that NT5E protein and mRNA levels are downregulated in stable NOTCH1-knockdown MDA-MB-231 cells; C and D: Online database prediction indicated a positive correlation between the mRNA expression levels of NOTCH1 and NT5E in breast cancer; E: In the 101 triple-negative breast cancer patients in The Cancer Genome Atlas, the mRNA expression levels of NOTCH1 and NT5E were positively correlated.; F-I: Online database prediction suggesting that low expression of NOTCH1 or NT5E was associated with better overall survival and disease-free survival in patients. ^aP < 0.05; ^bP < 0.01.

Figure 4 Knockdown of NOTCH1 decreases NT5E to inhibit breast cancer proliferation and migration both in vitro and in vivo. A: Western blot results validating the efficiency of stable NOTCH1 knockdown combined with stable NT5E overexpression in MDA-MB-231 cells; B and C: CCK-8 and colony formation assay showing that overexpression of NT5E reversed the inhibitory effect of NOTCH1 knockdown on breast cancer cell proliferation and colony formation; D and E: Transwell and scratch assays demonstrating that NT5E overexpression can reverse the inhibitory effect of NOTCH1 knockdown on breast cancer cell invasion and wound healing; F-H: Mouse xenograft tumor model showing that NT5E overexpression reversed the inhibitory effect of NOTCH1 knockdown on triple-negative breast cancer tumorigenesis in vivo. ^aP < 0.05; ^bP < 0.01; ^cP < 0.001.

Figure 5 Knockdown of NOTCH1 decreases NT5E to enhance the chemosensitivity of triple-negative breast cancer cells to cisplatin. A: Cisplatin IC₅₀ value analysis showing that re-expression of NT5E in NOTCH1-knockdown cells increased the resistance to cisplatin (IC₅₀ = 17.35 μM); B: CCK-8 assay showing that knockdown of NOTCH1 combined with cisplatin treatment inhibits cell proliferation, but this effect is reversed by NT5E overexpression; C: TUNEL assay showing that knockdown of NOTCH1 combined with cisplatin treatment significantly increases the apoptosis of cells, but this effect is reversed by NT5E overexpression. ^aP < 0.001.