Journal: Nature Communications

Article Title: FAD synthase confers ferroptosis resistance and restrains CD8 + T cell recruitment in hepatocellular carcinoma

doi: 10.1038/s41467-025-64572-y

Figure Lengend Snippet: a Comparisons of VB2 metabolism scores between tumors and non-tumor livers from TCGA-LIHC, OEP000321, and ICGC-LIRI-JP datasets. Scores were calculated for each sample using the ssGSEA algorithm based on a VB2 metabolism gene signature. b Kaplan-Meier overall survival curves stratified by VB2 metabolism scores, with optimal cutoffs determined by the surv_cutpoint function. c t-SNE visualization showing 7 major cell types in tumor samples from the immunotherapy cohort ( n = 8). d Proportions of the 7 major cell types across the eight patients. e Box plots with medians (red line) and interquartile ranges depicting VB2 metabolism scores in various cell types. Scores were calculated with the AUCell algorithm based on the VB2 metabolism gene signature. f UMAP plot showing 12 major cell types in the GSE202642 dataset. g Violin plot showing VB2 metabolism scores among tumor cells and normal epithelial cells in GSE202642 . h Schematic diagram of the VB2 metabolic pathway with metabolites in black and enzymes in red. i Hep3B and HCCLM3 cells treated with VB2 (10 µM), FMN (10 µM), FAD (10 µM), or PBS (Ctrl) for 48 h, followed by CCK-8 assay ( n = 5 biological replicates). j Subcutaneous tumors were established in Rag1 -/- mice with Hepa1-6 cells ( n = 5 per group). Mice received daily intratumoral injections of VB2 (50 mg/kg) or control solvent from day 5 to day 21. Tumor volumes were measured at the indicated timepoints. Tumors were photographed and weighed at day 21. k Proliferation of Hep3B and HCCLM3 cells with lentiviral knockdown of RFK (sh RFK ) or FADS (sh FLAD1 ) ( n = 5 biological replicates), as determined by CCK-8 assay and colony formation assay. l Migration of HCCLM3 cells with RFK or FADS knockdown, assessed by transwell assay ( n = 3 biological replicates). For bar graphs, data are presented as mean ± SD ( i – k ). For box plots ( a , e ), the box shows the median with interquartile ranges (IQR), whiskers extend to 1.5 × IQR, and outliers are shown as individual points. P- values were calculated using one-way ANOVA with Tukey’s test ( i , k ), two-way ANOVA with Tukey’s test ( j , k ), unpaired two-sided Student’s t test ( j ), two-sided Wilcoxon rank-sum test ( a , e , g ), and log-rank test ( b ). Source data are provided as a Source Data file.

Article Snippet: Antibody information: β-actin (CST, #4970, 1:1000), RFK (Santa Cruz, #sc-398830, 1:500), FADS (Proteintech, #68491-1-Ig, 1:5000), PCBP2 (ABclonal, #A23987, 1:2000), FLAG-tag (ABclonal, #AE005, 1:10000), His-tag (ABclonal, #AE003, 1:5000), HA-tag (ABclonal, #AE008, 1:2000), USP10 (Proteintech, #19374-1-AP, 1:1000), cGAS (Proteintech, #26416-1-AP, 1:2000), STING (Proteintech, #80365-RR, 1:1000), TBK1 (CST, #3504, 1:1000), phospho-TBK1 (CST, #5483, 1:1000), IRF3 (Proteintech, #11312-1, 1:5000), and phospho-IRF3 (CST, #29047, 1:1000).

Techniques: CCK-8 Assay, Control, Solvent, Knockdown, Colony Assay, Migration, Transwell Assay