Fig. 4.

In vitro ferroptosis evaluation. A) ROS generation assay of C4-2B_Enz cells treated with t-ML@DGLA/siDECR1 and other control groups (DGLA: 100 ​μg/mL, siRNA: 10 ​nM, bars: 50 ​μm), 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) was used as a probe; B) mitochondrial potential depolarization assay (DGLA: 100 ​μg/mL, siRNA: 10 ​nM, bars: 50 ​μm), rhodamine 123 (Rho 123) was used as a probe; C,D) Intracellular Fe content of C4–2B or C4-2B_Enz cells was detected by an intracellular iron colorimetric assay kit (OA@Fe₃O₄: 10 ​μM, DGLA: 25 ​μg/mL, siRNA: 2.5 ​nM, n ​= ​3, mean ​± ​SD, one-way ANOVA, n.s.: no significance, ∗∗P ​< ​0.01, ∗∗∗P ​< ​0.001, ∗∗∗∗P ​< ​0.0001); E,F) Relative MDA content of C4–2B or C4-2B_Enz cells was detected by a Lipid Peroxidation (MDA) Assay kit (OA@Fe₃O₄: 10 ​μM, DGLA: 25 ​μg/mL, siRNA: 2.5 ​nM, n ​= ​3, mean ​± ​SD, one-way ANOVA, ∗P ​< ​0.05, ∗∗P ​< ​0.01, ∗∗∗P ​< ​0.001, ∗∗∗∗P ​< ​0.0001); G) QF-PCR analysis of DECR1 and GPX4 mRNA levels (DGLA: 100 ​μg/mL, siRNA: 10 ​nM, n ​= ​3, mean ​± ​SD, multiple t-test, ∗∗∗∗P ​< ​0.0001); H) Western blot analysis of DECR1 and GPX4 protein levels (DGLA: 100 ​μg/mL, siRNA: 10 ​nM).