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. 2020 Sep 14;11:4591. doi: 10.1038/s41467-020-18245-7

Fig. 3. Radiation-induced CD47 transcription is regulated via HER2–NF-κB axis.

Fig. 3

Radiation enhanced CD47+ cell populations were reduced by inhibition of HER2 in MCF7/C6 (a) and in SKBR3 cells (b) treated with Lapatinib (10 µM, 72 h) in the presence or absence of IR (5 Gy) (n = 3; *P < 0.05, **P < 0.01, ***P < 0.001). c Immunoblotting of CD47 in HER2+ RD-BCSCs treated with PBS or Herceptin (10 µg/ml for 5 days refreshed on 2nd and 4th day) followed by sham or 5 Gy IR (HER2−RD-BCSCs included as a control). d CD47+ populations detected by flow cytometry in HER2+ RD-BCSCs and HER2+ RD-BCSCs with CRISPR-mediated knockout of HER2 (HER2−/−), n = 3; **P < 0.01. e Basal and radiation-induced CD47 expression was absent in HER2+ RD-BCSCs with CRISPR-mediated knockout of HER2 (HER2−/−). f NF-κB luciferase reporter activity in radioresistant MCF/C6 cells and MCF/C6 cells with HER2−/−, n = 3, ***P < 0.001. g CD47 promoter-controlled luciferase reporter activity containing wild-type or mutant NF-κB-binding motif in MCF7/C6 cells treated with TNFα (n = 3, ***P < 0.001). h CD47 promoter activity with wild-type or mutant NF-κB-binding motif measured in control and irradiated MCF7/C6 cells in the presence or absence of NF-κB inhibitor IMD-0354 (2 μM, 5 h; n = 3, **P < 0.01, ***P < 0.001). i Representative images of immunofluorescence of CD47 in irradiated MCF7 and MCF7/C6 cells pretreated with IMD-0354 (IMD); CD47 was visualized by confocal microscope 16 h after IR. Scale bar, 25 µm. j Immunoblotting of CD47 and HER2 in IR-treated MCF7/C6 cells with or without IMD-0354. k ChIP-qPCR assay for NF-κB recruitment in human CD47 promoter in MCF7/C6 cells irradiated (5 Gy) or treated with 10 ng/ml TNF-α. Chromatin precipitation was conducted with anti-p65 where anti-c-Rel served as a positive control in IκB-α promoter, IgG served as a negative control. l Reduced HER2-expressing populations in MCF7/C6 cells with CRISPR/Cas9-mediated knockout of CD47.