Fig 1: SLC7A11 expression is correlated with sorafenib resistance in HCC cells. A Expression of ferroptosis-related genes detected in parental cells and SR cells. B and C SLC7A11 expression level in parental and SR cells as determined by western blot analysis. D and E SLC7A11 expression levels in parental and SR cells as determined by IF analysis. F Volcano plot showing differentially expressed genes between the parental and resistant cells in the GSE158458 gene set, where SLC7A11 is highly expressed in both sorafenib resistant Huh7 and Hep3B cells. *p < 0.05; **p < 0.01; ***p < 0.001
Fig 2: A and B Volume of subcutaneous tumors in mice of different treatment groups. C Tumor weight of subcutaneous tumors in mice of different treatment groups. D and E Area of death and Ki-67-positive cell ratio of subcutaneous tumors in the four groups of mice. F Expression levels of FASN, HIF1a, and SLC7A11 in different treatment groups as determined by IHC analysis. G Schematic illustration demonstrating that FASN binding to HIF1a facilitated the entry of HIF1a into the nucleus to promote SLC7A11 transcription thereby inhibiting ferroptosis and promoting sorafenib resistance, while orlistat reversed sorafenib resistance by inhibiting FASN expression. Scale bars, 100 µm. *p < 0.05; **p < 0.01; ***p < 0.001. sora, sorafenib; orli, orlistat; Ferro-1, ferrostatin-1
Fig 3: Extracellular vesicles derived from PKM2-activated T lymphocytes promote lipid peroxidation in macrophages.Equal concentrations (10 µg/mL) of EVs from the PKM2fl/fl-C, PKM2fl/fl-Hcy, LckCrePKM2fl/fl-C or LckCrePKM2fl/fl-Hcy T lymphocytes were cultured with RAW264.7 cells for 24 h n = 3–5. Quantification of intracellular lipid peroxidation (LPO) (A) and malondialdehyde (MDA) (B). (C) Intracellular ROS levels in RAW264.7 cells were measured by a DCFH-DA probe through flow cytometric analysis and quantification. (D) Flow cytometric analysis of BODIPY 581/591 C11 and quantification of the oxidized BODIPY-C11 (emission: 590 nm)/reduced BODIPY-C11 (emission: 510 nm) ratio in RAW264.7 cells. Measurement of the antioxidant GSH (E) and NADPH/NADP+ ratio (F) in the RAW264.7 cells treated with different EVs. (G) Protein expression of Gpx4 and Slc7a11 was measured via Western blots, and the Gpx4 and Slc7a11 protein levels (relative to ß-actin or GAPDH) in RAW264.7 cells was quantified after EV treatment for 48 h. (H) Gene expression levels of Gpx4, Slc7a11, Ptgs2, SOD1 and Cat were measured via qPCR in RAW264.7 cells at 24 h after treatment with EVs. (I) Transwell migration assays of EV (10 µg/mL)-treated peritoneal macrophages in the upper chamber that migrated toward MCP-1 (20 ng/mL) in lower chamber with or without the lipid peroxidation inhibitors ferropstatin-1 (Fer-1, 5 µmol/L) or liproxstatin-1 (Lip-1, 1 µmol/L). Representative images of crystal violet staining were captured at 48 h after incubation to indicate migrated cells, and quantification of the migrated cells is shown. Cells were counted from 5 random microscope fields for each sample in 5 independent experiments. The data are presented as the mean ± SEM. (A–H) *p < 0.05, compared with PKM2fl/fl-C. #p < 0.05, compared with LckCrePKM2fl/fl-C. ?p<0.05, compared with PKM2fl/fl-Hcy. The data were compared using one-way ANOVA followed by Tukey's multiple comparison test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig 4: A Correlations among HIF1α, FASN, and SLC7A11 based on the TCGA database. B and C HIF1α protein expression in FASN-knockdown and control Huh7SR and 7721SR cells. D Sublocalization of FASN and HIF1α in the cytoplasm in Huh7SR and 7721SR cells under normoxic and hypoxic conditions as determined by IF analysis. E Detection of binding between FASN and HIF1α by immunoprecipitation under normoxic and hypoxic conditions. F Schematic representation of the HIF1α recognition motif obtained from the JASPAR database (top panel) and three fragments of the SLC7A11 promoter sequence (bottom panel). G The pGL4.10- SLC7A11 promoter reporter gene was co-expressed with HIF1α in HEK293T cells for 24 h. Luciferase expression was measured and normalized to Renilla. H Schematic diagram of HIF1α binding site of the SLC7A11 promoter and the binding sequence. I and J HIF1α and SLC7A11 protein expression in HIF1α-overexpression and control Huh7SR and 7721SR cells. K and L Effect of YC-1 on FASN, HIF1α and SLC7A11 protein expression in Huh7SR and 7721SR cells overexpressing FASN. *p < 0.05; **p < 0.01; ***p < 0.001. sora, sorafenib; Z-VAD, Z-VAD-FMK; Ferro1, ferrostatin-1; Necro-1, necrostatin-1
Fig 5: A GSEA enrichment analysis of FASN-related genes based on the TCGA database. B Proliferation of sorafenib-exposed FASN-knockdown and control Huh7SR cells treated with different cell death inhibitors as determined with the CCK-8 assay. C and D Detection of lipid ROS levels in FASN-knockdown and control Huh7SR and 7721SR cells exposed to sorafenib as determined by flow cytometry. E MDA levels in FASN-knockdown and control Huh7SR and 7721SR cells exposed to sorafenib. F Expression levels of ferroptosis-related genes in FASN-knockdown and control Huh7SR and 7721SR cells as determined by RT-qPCR analysis. G and H SLC7A11 protein expression in FASN-knockdown and control Huh7SR and 7721SR cells. I and J Effect of SLC7A11 overexpression on lipid peroxidation levels in FASN-knockdown and control Huh7SR and 7721SR cells exposed to sorafenib. K Effect of SLC7A11 overexpression on MDA concentrations in FASN-knockdown and control Huh7SR and 7721SR cells exposed to sorafenib. *p < 0.05; **p < 0.01; ***p < 0.001. sora, sorafenib; Z-VAD, Z-VAD-FMK; Ferro1, ferrostatin-1; Necro-1, necrostatin-1
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