Fig 2: Upregulation of ATP6V0D2 by ATL-I accelerated the autophagic degradation of EPAS1. (A) expression detection of HIF1A, EPAS1, VEGFA, FLT1/VEGFR1, KDR/VEGFR2, and FLT4/VEGFR3 by rt-qPCR in 786O and OSRC2 cells treated with 0, 80, and 160 μM ATL-I for 48 h. (B) Western blot was performed to detect the protein levels of HIF1A, EPAS1 and VEGFA in 786O and OSRC2 cells that were treated with 0, 80, or 160 μM ATL-I for 72 h. (C) 786O and OSRC2 cells were untreated or treated with 80 μM ATL-I for 48 h. After washing with PBS, the cells were incubated with 20 µg/ml cycloheximide and collected at the indicated times. EPAS1 protein levels were determined by western blot. (D) ccRCC cells were untreated or treated with ATL-I for 48 h. Immunoblotting was performed to detect EPAS1 protein levels in the two groups of cells incubated with MG132 (20 μM) for the indicated times. (E) ccRCC cells were untreated or treated with ATL-I for 48 h. Immunoblotting was performed to detect EPAS1 protein levels in the two groups of cells that were not incubated or incubated with chloroquine (10 μM) for 12 h. (F) Heatmap showing the top 50 upregulated and downregulated genes between the control and ATL-I treatment groups. (G) protein levels of ATP6V0D2 in 786O and OSRC2 cells treated with different concentrations of ATL-I. (H) immunoblotting was performed to detect the expression of EPAS1 and VEGFA in sh-nc and sh-ATP6V0D2 ccRCC cells with or without ATL-I treatment. (I) 786O cells with an expression vector or ATP6V0D2 knockdown were treated with 20 µg/ml cycloheximide at the indicated times. EPAS1 and ATP6V0D2 protein levels were determined by immunoblotting. (J) 786O cells with an expression vector or ATP6V0D2 knockdown were treated with 10 μM chloroquine or 20 μM MG132 for 8 h. EPAS1 and ATP6V0D2 protein levels were detected by western blot. (K) 786O cells, expression vector or knockdown of ATP6V0D2, were unincubated or incubated with 20 μM MG132 for 6 h. The cells were stained with anti-EPAS1/HIF2α and LysoTracker. The colocalization of EPAS1 and lysosomes in the cytoplasm was quantified. Scale bar: 10 μm. (*p < 0.05, **p < 0.01, ***p < 0.001.).

Fig 3: ATL-I enhances sunitinib sensitivity in ccRCC via inhibition of the EPAS1 pathway. (A) wildtype or sunitinib-resistant cells were treated with different concentrations of sunitinib for 48 h, and cell viability was measured by CCK-8 assay. (B) immunoblot analysis of EPAS1, VEGFA, and ATP6V0D2 in the indicated cells treated with or without sunitinib. (C and D) the indicated cells were treated with 5 μM sunitinib alone or in combination with 80 μM ATL-I for 36 h. Cell viability and apoptosis were determined by CCK-8 and flow cytometry assays. (E and F) 786O or SU-R-786O cells (1 × 106) were injected subcutaneously into nude mice. The indicated five groups of mice were either treated with sunitinib (20 mg/kg) alone or treated with ATL-I (50 mg/kg) and sunitinib (20 mg/kg). Then, the mice were sacrificed, and the tumor volume was measured. (G) IHC staining showing the expression levels of MKI67, PECAM1, and EPAS1. Scale bar: 50 or 100 μm; TUNEL assay for cell apoptosis analysis in the five groups. Scale bar: 50 μm. (**p < 0.01, ***p < 0.001.).

Fig 4: 5-HMF upregulates IL-22 receptor expression through HIF-dependent transcriptional activation. (A, B) expression of IL-22 receptor subunits in intestinal crypts. (A) mRNA and (B) protein levels of IL22R1 and IL10R2 in mice treated with vehicle, low-dose (50 mg/kg), or high-dose (100 mg/kg) 5-HMF. (C) Bioinformatics analysis of the IL22R1 promoter region. The schematic illustrates the positions of four putative hypoxia response elements (HREs; consensus core sequence) within the murine IL22R1 locus. Supporting evidence from publicly available human ChIP-seq data (GEO database) is shown below. The track labeled “Batch4_CHROM1_LOVO_EPAS1_RABBIT” visualizes HIF-2α (encoded by EPAS1) binding sites on chromosome 1 in the LOVO human colon epithelial cell line (experimental batch 4; rabbit antibody). The prominent peak (red box) indicates significant enrichment of HIF-2α binding within the upstream regulatory region of the human IL22R1 gene, supporting its status as a direct transcriptional target under HIF-mediated regulation. (D) mouse IL22R1 promoter scheme showing distal (−1525~-1449 bp) and proximal (+36 ~ +74 bp) HERs identified by sequence analysis. (E) dual-luciferase reporter assay in HEK-293 cells with an IL22R1 promoter-luciferase construct containing a wild-type sequence (−2000 wt) or mutations of distal or/and proximal HERs with transactivation by HIF-1, HIF-2, or GFP-control plasmids (n = 3 transfections/group). Asterisks (*) indicate the comparison of HIF1 or HIF2 to their respective GFP controls. ** p < 0.01, *p < 0.001 and NS, no significant difference. The cross (†) indicates a comparison of the transactivation of the wild-type sequence to the sequences mutated by HIF1 (†) or by HIF2 (†). † p < 0.05. All the error bars represent the means±sds