Fig. 7

Polyamine Supplementation Alleviates FMP-Induced Ferroptosis in 16HBE Cells. 16HBE cells were pretreated with spermine (SP, 50 µM) or spermidine (SPD, 100 µM), followed by 48-hour exposure to FMP (20 µg/ml). Cell death was assessed with Hoechst 33,342/PI staining. Intracellular SP/SPD levels and ROS levels, along with the protein expression levels of ALOX15, ALOX12, HO1 and GPX4 were assessed. Data were expressed as mean ± SD, N = 3 per group; * indicates significant difference from the blank control group (p < 0.05); ** indicates significant difference from the blank control group (p < 0.01); # indicates significant difference from 20 µg/ml FMP group (p < 0.05); ## indicates significant difference from 20 µg/ml FMP group (p < 0.01). (A) Cell death ratio in 16HBE cells following 48 h exposure to FMP (20 µg/ml) with or without pretreatment with 50 µM SP or 100 µM SPD for 4 h (N = 3). The blue and red dots indicated cells marked with negative (low) PI staining or positive (high) PI staining. (B) ELISA quantification of intracellular SP levels in 16HBE cells following 48 h exposure to FMP (20 µg/ml) with or without pretreatment with 50 µM SP or 100 µM SPD for 4 h (N = 5). And ELISA quantification of intracellular SPD levels in 16HBE cells (N = 5). (C)Quantification of intracellular Fe2 + levels in 16HBE cells following 48 h exposure to FMP (20 µg/ml) with or without pretreatment with 50 µM SP or 100 µM SPD for 2 h (N = 3). (D)Quantification of intracellular MDA levels in 16HBE cells following 48 h exposure to FMP (20 µg/ml) with or without pretreatment with 50 µM SP or 100 µM SPD for 2 h. Cells were treated with 100 µM hydrogen peroxide for 4 h as a positive control(N = 4). (E) Quantification of intracellular ROS levels in 16HBE cells following 48 h exposure to FMP (20 µg/ml) with or without pretreatment with 50 µM SP or 100 µM SPD for 2 h. Cells were treated with 50 µg/ml ROSUP for 2 h as a positive control (N = 5). (F) Representative western blotting images for ALOX15, ALOX12, HO1 and GPX4 in 16HBE cells following 48 h exposure to FMP (20 µg/ml) with or without pretreatment with 50 µM SP or 100 µM SPD for 2 h. (G) Semi-quantification of ALOX15, ALOX12, HO1 and GPX4 protein expression levels (N = 4)