Fig. 6
Naringenin mediates the process of superoxide stimulating PKC-ζ activity via the release of Zinc from Zinc finger domain of PKC-ζ. A The multiple sequence alignments of different PKC isoforms were carried out using Clustal Omega. The location of the first zinc finger motif is highlighted in green while the second zinc finger motif is highlighted in deep red. The solely zinc finger motif in atypical PKCs is conserved with the second in both classical and novel PKCs, and also highlighted in deep red. B Number of cysteines in the whole protein or the zinc finger motif of the cysteine-rich region. C PRKCZ and PRKCI mRNA relative expressions in 4T1 cells. Total mRNA was extracted and reverse transcribed into cDNA. Quantitative PCR was used to detect the transcriptional expression levels of PRKCZ and PRKCI genes to β-Actin gene. D, E Naringenin reduces the elevated superoxide (O2.) induced by radiation or paraquat. 4T1 cells were incubated with Dihydroethidium (DHE, 5 µM) for 30 min prior to treatment by radiation (X-ray, 8 Gy). Naringenin (Nar, 100 µM, 200 µM) was added to 4T1 cells for indicated time and MFI of DHC of cells was detected by flow cytometry (D). Paraquat (PQ, 100 µM) with or without naringenin (Nar, 100 µM) were added to 4T1 cells for 120 min and MFI of DHC of cells was detected by flow cytometry (E). F Naringenin inhibits the release of Zinc from 4T1 cells induced by radiation. 4T1 cells were treated with Nar (200 µM) for 30 min. X-Ray of 2, 4 or 8 Gy dose was used to treat 4T1 cells specifically. Cells were stained for 1 min with 150 nM of TSQ dissolved in Lock’s buffer (pH 7.4) and were examined under a fluorescence microscope. G, H Western blot of total protein lysate from cells treated with paraquat (PQ, 100 μM) and superoxide dismutase (SOD, 500 U/mL) or naringenin (Nar, 200 µM). I Model of Naringenin on TGF-β1EV release. Naringenin decreases TGF-β1EV release via the superoxide-Zinc-PKC-ζ-TGF-β1EV release pathway. Following ① radiation inducing superoxide elevation, ② Zinc is released Zinc from the finger domain of PKC-ζ and then PKC-ζ is phosphorylated, ③ which increases the number of releasable vesicles associated with TGF-β1, ④ promotes CD4+ T cells uptake, ⑤ activates TGF-β1 signaling, and ⑥ induces Treg cells infiltration and potential radiotherapy resistance. **p < 0.01; ***p < 0.001; ****p < 0.0001. Data were analyzed by t-test