Fig 2: TAM treated with DHA showed prominent DNA damage and activation of NF-?B in vitro and in vivo. (A,B) The DNA double-strand break was detected through the comet assay. The comet tail length was counted. (C–E) The expression of p53 and ?-H2A.X, which are biomarkers of the DNA damage response, was investigated using WB. The mean gray was quantitatively assayed. (F) The nuclear-translocation of NF-?B was observed by immunofluorescent staining. Green fluorescence came from NF-?B. (G–I) WB results revealed the phosphorylation of NF-?B, as indicated by the enhanced expression of p-NF-?B. NF-?B expression was also measured as control. The mean gray was quantitatively assayed. Values were means ± SD (n = 3, *p < 0.05 which was compared with the control group).

Fig 3: DHA@MIL-101 NRs facilitated DNA and mitochondria damage of Lewis cells. A Schematic illustration of DNA and mitochondria damage by DHA@MIL-101 NRs sourced ROS and LPO. B, C Lewis cells were incubated with DHA, MIL-101 NRs and DHA@MIL-101 NRs respectively. Comet experiments were applied to detect the DNA double-strand breaks (DDSB). The tail moment length was analyzed quantitatively via randomly selected cells. D The expression of p53 and ?-H2A.X, which are biomarkers of DNA damage, was assayed by WB. E The expression of cGAS, STING, which indicates DNA damage response (DDR), was assayed by WB. F The nuclear-translocation of NF-?B, which is direct evidence of NF-?B activation, was assayed by immunofluorescent staining. G Mitochondrial membrane potential was detected using a JC-1 probe with confocal microscopy. H The morphology of mitochondria was observed with TEM. All values are means ± SD (n = 3, * p < 0.05)

Fig 4: PDT induced by Ce6 led to DNA damage in vivo. p53 and ?-H2A.X expressions, which indicated the activation of DDR, were analyzed by IHC staining. Moreover, the expression of Bax and caspase-3 was also detected by IHC assay.

Fig 5: Inhibition of ferroptosis using Fer-1 reduced the DNA damage and activation of NF-?B triggered by DHA. (A,B) The LPO generation, which reflected the ferroptosis of TAM, was assayed using the C11-BODIPY probe. The cellular LPO fluorescence (FITC) was measured through flow cytometry. (C,D) The DDSB was assayed by comet experiments. The comet tail length was counted. (E–G) The expression of p53 and ?-H2A.X, and comet assay were detected to confirm the degree of DNA damage. The results were quantitatively analyzed using mean gray. (H–J) Activation of NF-?B was assayed through the expression of p-NF-?B in TAM. The results were quantitatively analyzed using the mean gray. (K) The LPO generation, which was assayed using the C11-BODIPY probe, was observed by confocal microscopy. Green fluorescence represented LPO accumulation. Values were means ± SD (n = 3, *p < 0.05).