Fig 2: Methylation of G3BP2 by PRMT5 confers lipid metabolism reprogramming.A The KEGG pathway analysis of the lipid metabolism-associated pathway affected by G3BP2 in Tu212 cells. B The KEGG pathway enrichment analysis of G3BP2-conferred metabolism pathways in Tu212 cells. C The effect of G3BP2-WT or G3BP2-R468K on the expression of lipid metabolism-associated genes were measured by RT-PCR analysis in Tu212 cells. D The effect of G3BP2-WT and G3BP2-R468K on the lipid metabolism-associated proteins were measured by immunoblotting analysis. E ACLY and FASN luciferase activity were determined by co-transfected with PRMT5-WT/PRMT5-MUT and pSV-Renilla to Tu686 cells. Luciferase activities were measured 60 h later. ACSL3 gene was used as an negative control. F, G The G3BP2-WT or G3BP2-R468K along with ACLY and FASN luciferase reporter plasmids were co-transfected into HEK293 or Tu686-PRMT5(KO) cells for 60 h. The luciferase activities were analyzed by dual-luciferase reporter assay, and normalized to the activity of Renilla. The data are presented as the mean ± SD; ns no significant difference; *P < 0.05; ** P < 0.01,***P < 0.001.

Fig 3: LPCAT4 regulated ACSL3 expression via WNT/β-catenin/c-JUN signaling pathway. (A) GSEA analysis indicated that WNT signaling pathway was found to be significantly enriched in the high LPCAT4 expression group. (B) Western blot assay was used to examine protein expression level. (C) The three transcription factor-binding sites of c-JUN on potential ACSL3 promoter region were indicated. (D) RT-PCR was used to examine ACSL3 mRNA expression. (E) The ChIP assay was used to validate binding domains of c-JUN in the potential ACSL3 promoter. (F) The luciferase assay was used to confirm which binding sites were functional. (G, H) Western blot assay was used to examine protein expression level.

Fig 4: Lipoproteins carry multiple lipid species that could inhibit lipid peroxidation.a. Heatmap showing the number of doublings (log2) in 5 days of mouse (Gpx4) or human (GPX4) knockout cell lines in cell culture under the supplementation of PBS, vitamin E (Vit. E, 10 μM), vitamin K2 (Vit. K2, 10 μM), oleic acid (OA, 250 μM) or ferrostatin-1 (Fer-1, 1 μM).b. Number of doublings (log2) in 5 days of B16 and HY15549 Gpx4-KO and HeLa and 786-O GPX4-KO cells in vitro either untreated or supplemented with Fer-1 (1 μM), vitamin E (10 μM), vitamin K2 (10 μM), or OA (250 μM).c. Cellular lipid oxidation ratio using BODIPY-C11 in HeLa cells in the absence (gray) or presence (blue) of a GPX4 inhibitor (ML162, 125 nM) under supplementation or not of LDL (50 μg/mL), HDL (50 μg/mL), vitamin E (10 μM), vitamin K2 (10 μM), vitamin D3 (10 μM), vitamin A (10 μM), CoQ10 (10 μM), OA (250 μM) or Fer-1 (1 μM).d. Cellular lipid oxidation ratio using BODIPY-C11 in A-498 cells with intact GPX4 activity and under supplementation or not of LDL (50 μg/mL), HDL (50 μg/mL), vitamin E (10 μM), vitamin K2 (10 μM), OA (250 μM) or Fer-1 (1 μM).e. Scheme highlighting genes (light blue) that affect the cellular ability to utilize lipoprotein-transported lipids (dark blue) that inhibit lipid peroxidation.f. Immunoblot analysis of ACSL3 and AIFM2 in the indicated cell lines transduced with a sgControl, sgACSL3 or sgAIFM2. GAPDH is included as a loading control.g. Cellular lipid oxidation ratio using BODIPY-C11 in the indicated Karpas299 (left) or A-498 (right) cell lines transduced with sgACSL3 (light blue) or with a sgControl (grey) under GPX4 inhibition (ML162: 75 nM for Karpas299, 175 nM for A-498) and HDL supplementation (50 μg/mL).h. Cellular lipid oxidation ratio using BODIPY-C11 in the indicated Karpas299 (left) or A-498 (right) cell lines transduced with sgAIFM2 (dark blue) or with a sgControl (grey) under GPX4 inhibition (ML162: 75 nM for Karpas299, 175 nM for A-498) and HDL supplementation (50 μg/mL).b, Bars represent mean ± s.d.; c, d, g, h, bars represent median; b, c, d, g, h, n=3 biologically independent samples. Statistical significance determined by two-tailed unpaired t-tests as indicated or compared to untreated cells (b, d) or ML162 treated cells (c).

Fig 5: A schematic model that illustrates the present study’s findings. Circ_0124346 functions as a sponge for miR-223-3p, sequestering it within the cytoplasm and subsequently preventing its binding to the 3'-UTR of ACSL3 mRNA. Consequently, this interference results in increased ACSL3 expression. The increased expression of ACSL3 in PAAD cells enhances lipid metabolism, thereby promoting PAAD cell proliferation. However, in the absence of specific adsorption by circ_0124346, miR-223-3p binds to the 3'-UTR of ACSL3 mRNA, resulting in decreased ACSL3 expression. Consequently, lipid metabolism is attenuated in PAAD cells, inhibiting PAAD cell proliferation