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. 2023 Feb 13;62:102636. doi: 10.1016/j.redox.2023.102636

Fig. 3.

Fig. 3

Hemin-TLR4 signaling releases XO from AML12 cells. A) AML12 cells were treated with 10 μM hemin and media was collected between 0 min and 16 h after treatment for XO activity measurement using HPLC. B) AML12 cells were treated with scrambled or siTLR4 for 48 h prior to treatment with 10 μM hemin. Media was collected and XO activity was measured. C) AML12 cells were treated with the TLR4 inhibitor TAK242 for 30 min prior to treatment with 10 μM hemin. Media was collected and XO activity was measured. D) AML12 cells were treated with TAK242 for 30 min prior to treatment with 10 μM hemin or 2.5 μM oxyhemoglobin. Media was collected and measured for XO activity. AML12 cells were treated with TAK242 for 30 min prior to treatment with 10 μM hemin. Cellular XOR mRNA E) and protein F) were measured from the cells by qPCR and Western blot. $Values are mean ± SEM using a 1-way ANOVA with Dunnett multiple comparisons test. ∼Values are mean ± SEM using a 1-way ANOVA with Sidak's multiple comparisons test. XO, xanthine oxidase; AML, alpha murine liver; Scram, scrambled; TLR4, toll like receptor 4; OxyHb, oxyhemoglobin; XOR, xanthine oxidoreductase; HPLC, high performance liquid chromatography.