Fig. 2.

Effect of ATF4 binding to FSP1 promoters in MC3T3-E1 cells on ferroptosis. MC3T3-E1 cells were used to construct an in vitro osteoporosis model by treating them with D-galactose (25.5 mM) or without (control). The cells were then treated with Ferrostatin-1 (Fer-1). (A) ATF4 protein expression and quantification in MC3T3-E1 cells with and without Fer-1 treatment were assessed. (B) JASPAR prediction of ATF4 binding to the FSP1 promoter. (C) A ChIP assay confirmed the interaction between ATF4 and the FSP1 promoter in MC3T3-E1 cells. (D) ATF4 and FSP1 protein expression and quantification in shATF4- and shNC-transfected MC3T3-E1 and 293T cells were measured. (E) ChIP analysis of the interaction between ATF4 and the FSP1 promoter in shATF4- or shNC-transfected MC3T3-E1 cells. (F) Relative luciferase activity in ATF4-knockdown 293T and MC3T3-E1 cells transfected with either FSP1-WT or FSP1-Mut. (G) Quantification of ATF4 and FSP1 protein expression levels in MC3T3-E1 cells transfected with shFSP1 or ATF4 overexpression vectors, shFSP1 plus ATF4 overexpression vectors, or shNC plus NC. (H) ATF4 and FSP1 protein expression levels and quantification in high-glucose (HG)-treated MC3T3-E1 cells transfected with shFSP1 or ATF4 overexpression vectors, shFSP1 plus ATF4 overexpression vectors, or shNC plus NC. (I) Cellular Fe²⁺ concentrations in HG-treated MC3T3-E1 cells transfected with shFSP1 or ATF4 overexpressing vectors, shFSP1 plus ATF4 overexpressing vectors, or shNC plus NC. (J) Lipid ROS levels in HG-treated MC3T3-E1 cells transfected with shFSP1 or ATF4 overexpression vectors, shFSP1 plus ATF4 overexpression vectors, or shNC plus NC. TSS: Transcription Start Site. Input: positive control. WT: wild type. Mut: mutant. n = 3. Data are shown as mean ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001. FSP1, Ferroptosis suppressor protein 1; ROS, reactive oxygen species; ATF4, activating transcription factor 4; ChIP, chromatin immunoprecipitation