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. 2023 Mar 1;37(5-6):204–217. doi: 10.1101/gad.350211.122

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© 2023 Song et al.; Published by Cold Spring Harbor Laboratory Press

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Figure 4. — ALOX5 is required for HTTQ94-mediated ferroptosis induced by ROS stress and glutamate. (A) Cell death assay in HTTQ94 tet-on SK-N-BE(2)C cells with different ALOX knockdowns. Cells were transfected with control siRNA (ctrl) or ALOX family-specific siRNAs, followed by preincubation with 0.5 µg/mL doxycycline for 16 h and then treated with 350 μM TBH for 24 h. (B) qPCR analysis of the knockdown efficiency of ALOX family members in HTTQ94 tet-on SK-N-BE(2)C cells transfected with control siRNA or ALOX family-specific siRNAs. (C) Cell death assay in HTTQ94 tet-on HT-22 cells with ALOX5 knockdown. Cells were transfected with control siRNA (ctrl) or ALOX5-specific siRNA and then preincubated with 0.5 µg/mL doxycycline for 4 h, followed by 10 μM TBH treatment for 8 h. (Left panel) ALOX5 knockdown efficiency. (Right panel) Cell death assay. (D) Western blot analysis of ALOX5 and HTTQ94 in HTTQ94 tet-on SK-N-BE(2)C control Crispr and Alox5-Crispr cells treated with 0.5 μg/mL doxycycline for 16 h. (E) Cell death assay for mHTT tet-on SK-N-BE(2)C control Crispr and Alox5-Crispr cells. Cells were preincubated with 0.5 μg/mL doxycycline for 16 h, followed by incubation with 350 μM TBH for 24 h with/without 2 μM Ferr-1. (F) FACS analysis of lipid ROS production in HTTQ94 tet-on SK-N-BE(2)C control Crispr and Alox5-Crispr cells. Cells were preincubated with 0.5 μg/mL doxycycline for 16 h and then treated with 350 μM TBH for 6 h. Lipid ROS was stained with C11-BODIPY. (G) Cell death assay in HTTQ94 tet-on HT-22 cells. Cells were transfected with control siRNA (ctrl) or ALOX5 siRNA, followed by incubation with 0.5 μg/mL doxycycline and 10 mM glutamate for 16 h in the presence or absence of 2 μM Ferr-1. Cell deaths were calculated from three replicates. Data shown in A, C, E, and G are means ± SD. P-values were derived from two-tailed unpaired t-test. (***) P ≤ 0.001.