Fig 2: CircLMO1 promotes cervical cell ferroptosis, and inhibited cell proliferation and invasion in a miR-4291/ACSL4-dependent manner. Relative GSH content (A), MDA content (B), and ROS level (C) in CaSki cells were assessed after circLMO1 overexpression in the presence or absence of miR-4291 mimics (or siRNA-Acsl4). (D) CaSki cell proliferation were assessed using CCK-8 assay after circLMO1 overexpression in the presence or absence of miR-4291 mimics (or siRNA-Acsl4). (E, F) CaSki cell invasion were assessed using transwell invasion assay after circLMO1 overexpression in the presence or absence of miR-4291 mimics (or siRNA-Acsl4). **p < 0.01.

Fig 3: ACSL4 is a downstream target of the RB/E2F pathway.(A) E2F1 ChIP-Seq peaks on the ACSL4 gene in different cell lines. Twelve putative E2F1 binding sites are indicated at the top of the ChIP-Seq peak. Data were derived from E2F1 ChIP-Seq data sets generated from prostate cancer cell lines (GSE36614 and GSE67809) and HeLa (GSM935484) and MCF7 (GSM935477) cell lines. Note that ChIP-Seq traces show fold enrichment over input for E2F1 binding to ACSL4 in all cell lines. (B) Schematic map of E2F1 binding sites on the 3 kb human ACSL4 promoter and its full-length and 4 progressive deletion mutant luciferase constructs. (C) Luciferase reporter assay of full-length ACSL4 promoter in the absence or presence of E2F1, E2F2, or E2F3. (D) Immunoblot analysis of cell lysates from RB stable–knockdown PC3 cells transfected with EV, E2F1, E2F2, or E2F3 cDNA for 48 hours. (E) Luciferase reporter assay of ACSL4 3 kb full-length promoter and its progressive deletion mutants in the absence or presence of E2F1. (F) ChIP-qPCR of E2F1 binding to the linker region (negative control), cluster I, and cluster II on the promoter of ACSL4 and CDK1 loci (positive control) in DU145 cells. (G) ChIP-qPCR of E2F1 binding to cluster I and II on the promoter of ACSL4 in control or RB stable–knockdown LNCaP cells. (H) ChIP-qPCR of E2F3 binding to the linker region (negative control), cluster I, and cluster II on the promoter of ACSL4 and CDK1 loci (positive control) in DU145 cells. (I and J) Immunoblot analysis of cell lysates from control or RB stable–knockdown LNCaP (I) or PC3 (J) cells. (K) Immunoblot analysis of cell lysates from PC3 cells transfected with EV or RB1 cDNA for 48 hours. (L) Immunoblot analysis of cell lysates from PC3 cells with RB stable–knockdown or combined with E2F1 knockdown. (M–P) Analysis of correlation between the mRNA levels of ACSL4 and RB1 copy number alterations (M and N) or the mRNA levels of E2F3 (O) or E2F1 (P) in the Beltran et al. (40) or Kumar et al. (41) data set. In C and E, 1-way ANOVA with Tukey’s multiple-comparison test was used to determine significance. In F, G, and H, unpaired 2-tailed t test was used to determine significance. In M and N, Kruskal-Wallis with Dunn’s multiple-comparison test was used to determine significance. In O and P, Spearman’s correlation coefficient was used to examine the correlation (r, correlation coefficient). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Bar graphs in C, E–H, are mean ± SD from n = 3~4 biological replicates.

Fig 4: ACSL4 is critical to RB-regulated ferroptosis.(A) Bar graph showing the relative MS1 peak area intensity of all the identifiable AA-containing phospholipids in control or RB stable–knockdown LNCaP or PC3 cells. (B) Immunoblot analysis of cell lysates from of control or RB stable–knockdown PC3 cells in the absence or presence of ASCL4 stable knockdown. (C and D) Levels of lipid peroxidation after 6 hours of 1 µM RSL3 treatment (C) or cell viability assay after 24 hours of 50 nM RSL3 treatment (D) in control or RB stable–knockdown PC3 cells in the absence or presence of ACSL4 stable knockdown. (E and F) 24-hour dose-response curves of RSL3 treatment in control or RB1 stable–knockout LNCaP (E) or PC3 (F) cells pretreated with vehicle or 20 µM PRGL493 for 24 hours. In A, unpaired 2-tailed t test was used to determine significance. In C and D, 1-way ANOVA with Tukey’s multiple-comparison test was used to determine significance. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. All data are shown as the mean ± SD from n = 3~4 biological replicates.