Figure 4.
Trsp-deficient HSPCs are subject to ferroptosis. (A) Representative pictures showing colony formation of whole BM cells in methylcellulose using M3434 for myeloid colonies. Colonies were scored 7 days after plating in methylcellulose in M3434, M3436, and M3630, for myeloid, erythroid, and pre-B colonies, respectively (n = 3 independent experiments). (B) Schematic representation of programmed cell death pathways. Ferroptosis (left) is triggered by iron-dependent accumulation of lethal ROS and lipid peroxides. Ferroptosis can be inhibited by iron (Fe2+) chelators such as deferoxamine (DFO). α-Toc and Fer-1 inhibit ferroptosis by suppressing lipid peroxidation. Apoptosis and necroptosis (right) are primarily regulated via TNFR1 signaling. Upon TNF binding, TNFR1 undergoes a conformational change, activating 2 potential cell death execution mechanisms. Caspase-8 triggers apoptosis by activating the classical caspase cascade and inactivating RIP1 and RIP3 through cleavage. As an alternative pathway, phosphorylated RIP1 and RIP3 initiate necroptosis independently of caspase-8. zVAD inhibits apoptosis by inhibiting caspase-8, whereas Necrostatin-1s (Nec-1s) inhibits necroptosis by inhibiting RIP1. (C) Relative MFI of C11-BODIPY 581/591 staining in Lin– cells from control mice and Trsp KO mice 24 hours after culturing in the presence of Fer-1 (1 μM; n = 5 independent experiments). (D) Frequency of DAPI– cells in Lin– cells measured 48 hours after treatment with Fer-1 (n = 5 independent experiments). (E) Heat map illustrating relative cell number of viable Lin– cells compared with day 0, as determined by the CellTiter-Glo Luminescent Cell Viability assay, 48 hours after culturing in the presence of Fer-1 (1-5 μM), α-Toc (1-2 mM), zVAD (25-50 μM), and Nec-1s (50-100 μM; n = 3 per group). (F) Colonies were scored 7 days after plating in methylcellulose (MethoCult M3434) in the presence of Fer-1 (5 μM) and α-Toc (2 mM; n = 3 per group). P values were calculated by a 2-sided Student t test or 1-way analysis of variance (ANOVA) test. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. BFU-E, burst-forming unit-erythroid; DMSO, dimethyl sulfoxide GSH, glutathione-SH; ns, not significant; RIP1, receptor-interacting serine/threonine-protein kinase 1; TNF, tumor necrosis factor; TNFR1, tumor necrosis factor receptor-1.

Trsp-deficient HSPCs are subject to ferroptosis. (A) Representative pictures showing colony formation of whole BM cells in methylcellulose using M3434 for myeloid colonies. Colonies were scored 7 days after plating in methylcellulose in M3434, M3436, and M3630, for myeloid, erythroid, and pre-B colonies, respectively (n = 3 independent experiments). (B) Schematic representation of programmed cell death pathways. Ferroptosis (left) is triggered by iron-dependent accumulation of lethal ROS and lipid peroxides. Ferroptosis can be inhibited by iron (Fe2+) chelators such as deferoxamine (DFO). α-Toc and Fer-1 inhibit ferroptosis by suppressing lipid peroxidation. Apoptosis and necroptosis (right) are primarily regulated via TNFR1 signaling. Upon TNF binding, TNFR1 undergoes a conformational change, activating 2 potential cell death execution mechanisms. Caspase-8 triggers apoptosis by activating the classical caspase cascade and inactivating RIP1 and RIP3 through cleavage. As an alternative pathway, phosphorylated RIP1 and RIP3 initiate necroptosis independently of caspase-8. zVAD inhibits apoptosis by inhibiting caspase-8, whereas Necrostatin-1s (Nec-1s) inhibits necroptosis by inhibiting RIP1. (C) Relative MFI of C11-BODIPY 581/591 staining in Lin– cells from control mice and Trsp KO mice 24 hours after culturing in the presence of Fer-1 (1 μM; n = 5 independent experiments). (D) Frequency of DAPI– cells in Lin– cells measured 48 hours after treatment with Fer-1 (n = 5 independent experiments). (E) Heat map illustrating relative cell number of viable Lin– cells compared with day 0, as determined by the CellTiter-Glo Luminescent Cell Viability assay, 48 hours after culturing in the presence of Fer-1 (1-5 μM), α-Toc (1-2 mM), zVAD (25-50 μM), and Nec-1s (50-100 μM; n = 3 per group). (F) Colonies were scored 7 days after plating in methylcellulose (MethoCult M3434) in the presence of Fer-1 (5 μM) and α-Toc (2 mM; n = 3 per group). P values were calculated by a 2-sided Student t test or 1-way analysis of variance (ANOVA) test. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. BFU-E, burst-forming unit-erythroid; DMSO, dimethyl sulfoxide GSH, glutathione-SH; ns, not significant; RIP1, receptor-interacting serine/threonine-protein kinase 1; TNF, tumor necrosis factor; TNFR1, tumor necrosis factor receptor-1.

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