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. 2022 Nov 28;30(2):369–382. doi: 10.1038/s41418-022-01089-7

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — A, B Western blots and quantification for changes of GCH1 in hSOD1^G93A cells transfected with different doses of SPY1-Flag (n = 3). C The effect of transfected SPY1-Flag and SiGCH1 on relative content of BH4 was shown (n = 6). D–G The effect of knockdown GCH1 on viability, lipid oxidation, and LIP in overexpressed SPY1 cells compared with control (in D and G, n = 6; in F, n = 3). Scale bar, 100 μM. H The viability was measured in SiSPY1 cells with transfected GCH1-Flag and treatment of BH4 (50 μM) and DFO (100 μM) for 24 h (n = 6). I The extent of lipid peroxidation was assessed by C11-BODIPY fluorescence staining followed by flow cytometry (channel 488). J The relative mRNA of TFR1, DMT1 and FPN1 in SPY1-Flag cells compared with control (n = 6). K Western blots for TFR1 in WT and hSOD1^G93A cells with overexpressed SPY1 or control were shown. L After sequential transfection with P53-HA and control and incubation with DFO (100 μM), Fer-1 (10 μM), Zvad (20 μM), and vehicle for 24 h. The viability of SPY1-Flag cells was assessed by CCK8 assay (n = 6). M Western blots for P53, TFR1, and GCH1 in SPY1-Flag cells with overexpressed P53 and control were shown. N Western blots were shown for expression of p-SP1, SP1, and GCH1 in SPY1-Flag cells with overexpressed SP1(S59A) and wild type. Values represent mean ± SD. Statistical analysis by one-way ANOVA followed by Tukey’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001.