Fig. 4

N-7DHC-lipos plus irradiation induces ferroptosis. N-7DHC-lipos were applied at a dose of 50 µg/mL. Except for clonogenicity studies, where a range of radiation doses were tested, irradiation (IR) was applied at 5 Gy. (a) Effect of the combination on cell viability, tested in H1299 cells using MTT assays (n = 5 biologically independent samples). N-CHOL-lipos plus IR was also tested for comparison. (b) Effect on clonogenicity, evaluated by clonogenic assay in H1299 cells. A range of radiation doses (0–10 Gy) was applied, together with N-7DHC-lipos or carrier only. Survival fractions relative to the unirradiated control were fitted to the linear-quadratic model (n = 3 biologically independent samples). (c) Summary of data fitting, based results from (b). α and β, fitting coefficients. SF₂ and SF₅, survival fractions at 2 and 5 Gy, respectively. D_10%, dose required to reduce the survival fraction to 10%. DMR, dose modifying ratio, based on SF_2,SF_{5, and}D_10%. (d) Viability of H1299 cells that were treated with N-7DHC-lipos plus IR, and co-incubated with cell death inhibitors, including Ferr-1, DFO, Z-VAD-FMK (Z-VAD), glycine (Gly), and 3-methyladenine (3-MA) (n = 5 biologically independent samples). (e) Levels of lipid peroxidation when H1299 cells were treated with N-7DHC-lipos and IR while co-incubated with or without Ferr-1, measured by TBARS assay (n = 3 biologically independent samples). (f) LDH release, measured in H1299 and Hcc827 cells (n = 5 biologically independent samples). Cells were treated with N-7DHC-lipos plus IR, while co-incubating with Ferr-1 or DFO. (g) ATP release from cells treated with N-7DHC-lipos plus IR, tested in H1299 cells (n = 5 biologically independent samples). (h) Clonogenic assay, tested in both H1299 and H460 cells. Cells were treated with N-7DHC-lipos plus IR, with or without co-incubation with Ferr-1 (n = 3 biologically independent samples). (i) Western blot analysis of ferroptosis-related markers, including KEAP1, GPX4, SLC7A11, SCL11A2, and ACSL4, in H1299 cells treated with N-7DHC-lipos and IR, alone or in combination. PBS, IR alone, and N-7DHC-lipos were tested for comparison. (j) (Left) TEM images of H1299 cells treated with N-7DHC-lipos plus IR. Red arrows point to mitochondria. Scale bars, 500 nm (upper) and 200 nm (lower). (Right) Bar graphs showing the percentage of normal and damaged mitochondria, based on TEM results. Compared to the untreated control (CTRL), many more damaged mitochondria were observed in cells treated with the combination of N-7DHC-lipos and IR, as manifested by shrunken size and condensed structure. (k) Schematic illustration of ferroptosis induced by N-7DHC-lipos plus IR. Data are presented as mean ± SD. Statistical difference was evaluated using one-way ANOVA in (a, d, and f-h), and two-tailed Student’s t-test in (b, e, and j). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001