Figure 5: NFKB1 knockdown reduces CD47 expression, decreases tumour size and increases phagocytosis of breast cancer cells. | Nature Communications

Figure 5: NFKB1 knockdown reduces CD47 expression, decreases tumour size and increases phagocytosis of breast cancer cells.

From: A CD47-associated super-enhancer links pro-inflammatory signalling to CD47 upregulation in breast cancer

Figure 5

(a) NFKB1 shRNA reduces the size of breast tumours derived from MCF7-Luc in xenotransplants (red dots). N=5 samples. Values represent mean±s.e.m. Student’s unpaired t-test for independent samples was performed. **P<0.01. Black dots: Control shRNA treated breast tumours derived from MCF7-Luc in xenotransplants. (b) Upper panels: Images of tumours 6 weeks after injection of MCF7 cells that were: untreated (control; left); infected with turboRFP-control shRNA (middle); or infected with turboRFP-NFKB1 shRNA (right). NFKB1 knockdown led to smaller tumour sizes. Scale bar: 500 μm. Lower panels: flow cytometry analysis of cells from dissociated tumours that had grown for 6 weeks. Left panel: TurboRFP (reporter) expression levels are similar in cells treated with turboRFP-control shRNA (yellow) versus turboRFP-NFKB1 shRNA (pink). Orange histogram shows overlap. Right panel: CD47 protein levels are lower on cells in tumours generated from MCF7 cells with NFKB1 knockdown (red) versus those without knockdown (controls with no shRNA (turquoise) or with control shRNA (orange)). CD47 expression is slightly higher in MCF7 cells infected with control shRNA than in uninfected MCF7, because any intracellular infection leads to an increased CD47 expression. Grey histograms are the FMO controls. (c) Phagocytic index of MCF7 cells is increased over control levels (empty vector lacking shRNA) by anti-CD47 blocking antibody (CD47α), NFKB1 shRNA infection, and more so by NFKB1 shRNA infection followed by anti-CD47 treatment. N=3 samples. Values represent mean±s.d. Student’s unpaired t-test for independent samples was performed. **P<0.0025 when compared to Control shRNA, ***P<0.0010, ****P<0.0005. (d) Inhibiting the binding of BRD4 to SEs by using JQ1 (1 μM) reduces CD47 expression over time in MCF7 breast cancer cells. N=3 samples. Values represent mean+s.d. Student’s unpaired t-test for independent samples was performed. *P<0.05, **P<0.01.

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