Fig 2: IRP1 targets Bcl-xL to modulate mitophagy. (A and B) HeLa cells with stable tetracycline (Tet)-induced Bcl-xL expression were pre-treated with Tet or the vehicle (V) for 6 h, and then incubated with DFO for 24 h (A) or transfected with si-ISCU or NC siRNA for 72 h (B). The cells were harvested and analyzed by immunoblotting. The relative levels of indicated bands are shown in the right histograms. Data represent the mean ± SEM of three independent experiments. **p < 0.01. (C and D) HeLa cells stably co-expressing mt-Keima and Tet-induced Bcl-xL were pre-treated with Tet or the vehicle (V) for 6 h, then incubated with DFO for 24 h (C) or transfected with si-ISCU or NC siRNA for 72 h (D). The cells were collected and signals from green and red channels were detected by flow cytometry. The percentage of mitophagic cells with higher red/green fluorescence was showed in the below histograms as mean ± SEM from three experiments; **p < 0.01. (E) HeLa cells stably expressed sc shRNA or sh-IRP1 were transiently transfected with si-Bcl-xL or NC siRNA. After 48 h of transfection, the cells were challenged with DFO for 24 h. The cells were harvested and analyzed by immunoblotting. The relative levels of TIM23 are shown in the right histograms. Data represent the mean ± SEM of three independent experiments. **p < 0.01. (F) HeLa cells stably expressed sc shRNA or sh-IRP1 were transiently transfected with si-Bcl-xL accompanied with si-ISCU or the NC siRNA. After 72 h of transfection, the cells were harvested for immunoblotting analysis. The relative levels of TOM20 are shown in the right histograms. Data represent the mean ± SEM of three independent experiments. *p < 0.05; **p < 0.01.

Fig 3: IRP2 is sufficient to sense ISC deficiency and sensitize cells to ferroptosis.(A) Proliferation (relative cell number) of IRP1/2 double-knockout MDA-MB-231 cells grown in media supplemented with 35 µM iron nitrate and 50 µM Trolox or upon withdrawal of supplements at day 0. (B) Immunoblots for indicated proteins of lysates derived from IRP1 knockout and IRP1/2 double-knockout MDA-MB-231 cell line treated with 50 µM DFO and 35 µM iron nitrate (1 day) as indicated. (B and C) Immunoblots for indicated proteins of lysates derived from parental (WT) and IRP1/2 double-knockout MDA-MB-231 cell line expressing a nontargeting shRNA (shGFP, -) or shRNA targeting NFS1 (shNFS1, +). (D) Relative abundance of TFR1 and FTH1 mRNA in parental, IRP1 knockout, and IRP1/2 double-knockout MDA-MB-231 cell line expressing shGFP or shNFS1. (E and F) Immunoblots for indicated proteins of lysates derived from IRP1/IRP2 double-knockout MDA-MB-231 cells expressing dox-repressible IRP2 treated with 100 nM dox for 2 days, and then treated with 100 nM dox, 50 µM DFO, or 35 µM iron nitrate (1 day), or infected with shGFP or shNFS1. After drug selection for shRNA transduction, cells are treated with 100 nM dox and 35uM iron nitrate for 1 day. (G) Relative viability of cells as in (E) and (F). After drug selection for shRNA transduction, cells were treated with 100 nM dox, 1 µM ferrostatin, or erastin (0, 2.5, 5, and 10 µM) for 2 days as indicated.

Fig 4: IRP1 is indispensable for iron stresses induced mitophagy. (A and B) HeLa cells stably expressed sc shRNA or sh-IRP1 were transiently transfected with mt-Keima. Following 24 h of expression, cells were treated with DFO for another 24 h (A), or transfected with si-ISCU for another 72 h (B), and then cells were imaged with 458 nm (shown in green) or 543 nm (shown in red) light excitation using a LSM 510 Zeiss confocal microscope. Representative images are presented. Scale bar, 10 µm. Graph at right showing the number of mt-Keima puncta per cell. Data represent the mean ± SEM of three independent experiments, each with >200 cells counted per condition. ***p < 0.001. (C and D) HeLa cells stably expressed sc shRNA or sh-IRP1 were transfected with WT IRP1, or C503S, R536Q mutant, then treated with DFO for another 24 h (C), or transfected with si-ISCU for another 72 h (D). The cells were then harvested and analyzed by immunoblotting. The relative levels of indicated bands are shown in the right histograms. Data represent the mean ± SEM of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001. (E and F) HeLa cells stably expressed sc shRNA or sh-IRP1 were exposed to 100 µM or 250 µM DFO for 12 h (E), or transfected with 10 pmol or 30 pmol si-ISCU or the NC siRNA for 48 h (F). Cells were harvested for blotting phosphorylated FUNDC1 level at Ser13 (F1–P13). The relative levels of F1–P13 to F1 are shown in the right histograms. Data represent the mean ± SEM of three independent experiments. Data represent the mean ± SEM of three independent experiments. ***p < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig 5: Traumatic brain injury (TBI)-induced damage of mitochondria and disruption of iron homeostasis were improved by ruthenium red (RR) in primary cultured neurons. (A, B) RR treatment significantly decreased the expression of Drp1, MIEF1 and Fis1 (A) and increased the expression of IRP1/2 (B) after TBI, however, Sper treatment had no obvious effect on these proteins. Data are presented as mean ± SEM, n = 6 per group; **P < 0.01, ***P < 0.001 vs control group; ## P < 0.01, ### P < 0.001 vs TBI + vehicle group; @ P > 0.05 vs TBI + vehicle group. ß-actin was used as a loading control