Fig 1: HCG regulates IL4I1 expression through polyamine metabolism. (A,B) Effects of different concentrations of HCG on the ODC1 protein level in Ishikawa cells. (C) PUT concentrations after Ishikawa cells were treated with 10 µg/mL HCG. (D,E) Western blot analysis of effects of PUT on IL4I1 expression in Ishikawa cells. (F) The effects of PUT on IL4I1 protein secretion in the supernatant of cultured Ishikawa cells. (G,H) Western blot analysis of the protein level of IL4I1 after Ishikawa cells were treated with HCG and DFMO. PUT, putrescine; DFMO, difluoromethylornithine. ns, no significance. *, p < 0.05; **, p < 0.01.
Fig 2: Survival analysis of tryptophan‐metabolizing enzymes in DLBCL and NK/TCL. Kaplan–Meier survival curves of (A) IDO1, IDO2, TDO2, and IL4I1 in DLBCL; (B) IDO1, TDO2, and IL4I1 in NK/TCL; (C) IDO1, IDO2, TDO2, and IL4I1 in TCGA cohort of DLBCL patients. DLBCL, diffuse large B‐cell lymphoma; NK/TCL, natural killer/T‐cell lymphoma.
Fig 3: IL4I1 promoted HCC survival after heat stress(A) RNA sequencing for MHCC-97H in 2D, 3D, and animal models (three biological replicates).(B) Upregulated genes in 3D and animal models, respectively, compared with the 2D model.(C) The KEGG enrichment of 95 overlapped genes.(D) Volcano plot of DEGs among “Metabolic pathways” in the 3D model versus 2D model. The names of 11 3D-specific metabolic genes are indicated in the plot.(E and F) qRT-PCR and western blot assay determining the IL4I1 expression of MHCC-97H in 2D and 3D models (three biological replicates).(G) Volcano plot of DEGs in HCC cells after heat stress compared with untreated control in the 3D model (three biological replicates). The names of 11 3D-specific metabolic genes are indicated in the plot.(H) Cell viability of MHCC-97H detected by trypan blue staining in different groups after heat treatments for 15 min (45°C, 47°C, and 49°C) (three biological replicates).(I and J) Representative images and quantification of the colony formation (4 × 103 cells/well), migration assay (1 × 105 cells/well), and invasion assay (1 × 105 cells/well) in IL4I1-overexpressed and control groups after 47°C treatment for 15 min (three biological replicates). Mean ± standard deviation. Statistical analysis, two-tailed Student’s t test for (E), (H), and (J). **p < 0.01, ***p < 0.001.
Fig 4: IL4I1-induced AHR activation promotes HCC survival after heat stress(A) Relative abundance of I3A and KynA in supernatants of Ctrl and IL4I1 MHCC-97H cells (4 days) (five biological replicates).(B) mRNA expression of IL4I1, IDO, and TDO2 in 2D and 3D models (three biological replicates).(C) Relative abundance of I3A and KynA in supernatants of MHCC-97H cells in the 2D and 3D models (4 days) (five biological replicates).(D) GSEA analysis of AHR pathway in the 3D model versus 2D model.(E) mRNA expression of AHR signature targets in 2D and 3D models (three biological replicates).(F) mRNA expression of CYP1A1 and CYP1B1 in IL4I1-overexpressed and control groups of MHCC-97H (three biological replicates).(G) Correlation between the expression of IL4I1 and AHR-targeted genes in TCGA HCC dataset.(H) Correlation between the expression of AHR and AHR-targeted genes in TCGA HCC dataset.(I) mRNA expression of CYP1A1 and CYP1B1 in MHCC-97H treated with I3A (50 µM) or KynA (250 µM) for 8 h (three biological replicates).(J and K) Representative images and quantification of the colony formation (4 × 103 cells/well), migration assay (1 × 105 cells/well), and invasion assay (1 × 105 cells/well) in MHCC-97H with or without I3A (50 µM) or KynA (250 µM) treatment for 8 h, following 47°C treatment for 15 min (three biological replicates). Mean ± standard deviation. Statistical analysis, two-tailed Student’s t test for (A), (B), (C), (E), and (F), one-way ANOVA with post Tukey’s comparisons test for (I) and (K). *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 5: Expression of tryptophan metabolizing enzymes using immunohistochemical staining in DLBCL and NK/TCL. (A) Representative images of IHC staining of IDO1, IDO2, TDO2, and IL4I1 in DLBCL and NK/TCL. (B) Analysis of the proportions of IDO1, IDO2, TDO2, and IL4I1 (negative, positive) in DLBCL and NK/TCL. DLBCL, diffuse large B-cell lymphoma; IHC, immunohistochemical; PD-L1, programmed death-ligand 1; NK/TCL, natural killer/T-cell lymphoma.
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