Fig. 5
Naringenin inhibited PKC-ζ to suppress TGF-β1EV secretion. A The phosphorylation level of PKC-ζ in 4T1 cells after treatment. 4T1 cells were treated by naringenin (Nar) in indicated concentration for 6 h followed by 8 Gy of X-Ray. The p-PKC-ζ level in cell lysate was detected by western blot. B The mRNA expression level of PKC-ζ. 4T1 cells were treated by naringenin in indicated concentration for 6 h followed by 8 Gy X-Ray. Total RNA was extracted and PKC-ζ mRNA expression was detected by qPCR 24 or 48 h after radiation. C, D 4T1 cells were treated same as A, B. EVs and EV-associated TGF-β1 were quantified by BCA (C) and ELISA (D), respectively. E Treg induced by EVs. 4T1 cells were treated by radiation with or without naringenin (200 µM) for 48 h and EVs in the culture medium were isolated and diluted with PBS in same volume. Naïve spleen cells from Foxp3-GFP mice were treated by anti-CD3/CD28 functional antibody with an equal volume of EVs for 72 h and CD4+GFP+ cells were analyzed by flow cytometry. F EV-associated TGF-β1 secreted by tumor. Tumor-bearing mice were treated by radiotherapy (10 Gy) with or without naringenin (100 mg/kg). Tumor tissues were dissected and cultured in vitro for 48 h. EVs were isolated and TGF-β1 in EVs was analyzed by ELISA. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. All experiments was analyzed by 2-way ANOVA