Three or more independent experiments were performed for all experiments. Animals and cell cultures for each group were randomized with the website www.randomization.com . Treatment, data collection, and data analyses were blinded by using different investigators or by masking sample labels.
For in vitro experiments, we tested different dosages of Hemin (0, 10, 50, or 100 μM) and RSL3 (0, 2, 4, 8, 16, or 32 μM) at 12 and 24 h respectively. We added 2 μM Ferrostatin-1 (Fer-1, S7243, Selleck), 100 μM Deferoxamine (DFO, Y0001937, Sigma), 100 μM Necrostatin-1 (Nec-1, N9037, Sigma), 1 mM 3-Methyladenine (3-MA, HY-19312, MCE, USA), or 10 μM Q-VD-OPh (SML0063, Sigma) at the same time with Hemin or RSL3 based on previously published work [21 (link), 48 (link)]. We treated cells with 20 μM t-BOOH (458139, Sigma, USA) for 12 h, with or without Fer-1 or 0.5 mM GSH (G4251, Sigma, USA) at the same time. Cells were collected for PI staining, TUNEL staining, immunofluorescence staining, flow cytometry, GPx4 activity assay, RT-qPCR, and RNA-seq.
For in vivo experiments, Fer-1 (2 mg/kg) or vehicle (0.1% DMSO (D1418, Sigma), 2.5% PEG300 (202371, Sigma) and 0.25% Tween80 (P1754, Sigma) in saline) was injected daily (i.p.) for the initial 7 days beginning 2 h after IVH. The assessment included behavior tests, neuroimaging, histology, and TEM.
For in vitro experiments, we tested different dosages of Hemin (0, 10, 50, or 100 μM) and RSL3 (0, 2, 4, 8, 16, or 32 μM) at 12 and 24 h respectively. We added 2 μM Ferrostatin-1 (Fer-1, S7243, Selleck), 100 μM Deferoxamine (DFO, Y0001937, Sigma), 100 μM Necrostatin-1 (Nec-1, N9037, Sigma), 1 mM 3-Methyladenine (3-MA, HY-19312, MCE, USA), or 10 μM Q-VD-OPh (SML0063, Sigma) at the same time with Hemin or RSL3 based on previously published work [21 (link), 48 (link)]. We treated cells with 20 μM t-BOOH (458139, Sigma, USA) for 12 h, with or without Fer-1 or 0.5 mM GSH (G4251, Sigma, USA) at the same time. Cells were collected for PI staining, TUNEL staining, immunofluorescence staining, flow cytometry, GPx4 activity assay, RT-qPCR, and RNA-seq.
For in vivo experiments, Fer-1 (2 mg/kg) or vehicle (0.1% DMSO (D1418, Sigma), 2.5% PEG300 (202371, Sigma) and 0.25% Tween80 (P1754, Sigma) in saline) was injected daily (i.p.) for the initial 7 days beginning 2 h after IVH. The assessment included behavior tests, neuroimaging, histology, and TEM.
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