Geyan Wu

Information on antibodies and reagents, including RRIDs

Supplementary figures and legends

Figure S11. Associations between SLC27A2 expression and various immune markers including PD-1, CTLA4, and TIM3

Figure S4. SLC27A2 deficiency specifically reduces LCFA uptake (A) Assessment of lipid accumulation changes following SLC27A2 modulation using triglyceride quantification. (B-D) FFA quantification was used to demonstrated that SLC27A2 specifically influences the uptake of LCFAs. Statistical significance was determined using Student’s t-test (A-D). *P < 0.05; **P < 0.01; ***P < 0.001. Abbreviations: OE, overexpression; KO, knockout; FFA, free fatty acid; C2:0, acetic acid (10 μM); C6:0, caproic acid (10 μM); C16:0, palmitic acid (10 μM).

Figure S18. PPAR-γ's role in SPP1 regulation by SLC27A2 (A-B) Analysis of PPAR-γ mRNA correlation with SPP1 and SLC27A2 in HCC. (C-E) PPAR-γ expression in THP-1-derived macrophages and BMDMs unaffected by FA chain lengths. (F) Changes in SPP1 mRNA levels post-PPAR-γ knockdown and co-culture with SLC27A2 KO or NC Hepa1-6 cells. (G) Quantitative analysis for Figure 7J. (H) Ki67 assay for figure 7J. Statistical techniques: one-way ANOVA with Tukey's test (C-D, and G-H) and Student’s t-test (F). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure S2. Hypoxia-induced transcriptional repression of SLC27A2 via HNF4A inhibition (A) Analysis of DNA copy number variations in SLC genes associated with HCC, using TCGA-LIHC data. (B) Assessment of methylation levels in the SLC27A2 promoter region in tumor versus non-tumor samples, derived from TCGA-LIHC. (C) Methodology for identifying transcriptional regulators of SLC27A2. (D) Correlation analysis of HNF4A, SLC27A2, and hypoxia markers in tumor samples from TCGA-LIHC. (E-F) Evaluation of HNF4A and SLC27A2 expression in HCC tissue samples through immunofluorescence, and their inter-relationship. (G) Correlation assessment of HNF4A and SLC27A2 in various HCC cell lines from the LIMORE database. (H) Schematic representation of HNF4A binding sites on the SLC27A2 promoter, confirmed by ChIP-PCR. (I-K) Luciferase reporter assays analyzed the effects of HNF4A overexpression (I) and hypoxia (J) on SLC27A2 promoter activity; mRNA levels were measured by qRT-PCR (K). Statistical significance was evaluated using the Wilcoxon t-test (B), Student’s t-test (H-J), Spearman (F), Pearson correlation (G), and one-way ANOVA followed by Tukey's test (K). *P < 0.05, **P < 0.01, ***P < 0.001. OE: overexpression; NC: negative control; CNV: copy number variation.

Figure S13. Associations between SLC27A2 expression and various immune markers and cell proportions in the context of Figure 5

Figure S5. Hypoxia attenuates FAO activity in HCC (A-B) Proteomic data (A) and single-cell RNA-seq data (B) from patients indicates downregulation of key enzymes responsible for LCFA β-oxidation in HCC, especially under hypoxia. (C) qRT-PCR was used to demonstrate the inhibition of FAO enzymes by hypoxia. Statistical significance was determined using Wilcoxon t-test (A-B), Student’s t-test (C).*P < 0.05, **P < 0.01, ***P < 0.001

Figure S6. Energy output and lipid peroxidation influenced by fatty acid chain lengths (A-B) Effects of palmitic acid concentrations on ATP production in HCC cells under normoxia (A) and hypoxia (B). (C-D) Glucose supplementation effects on ATP output under normoxia (C), comparing ATP differences between palmitic acid and glucose (D). (E-F) Impact of palmitic and glucose deprivation on ATP levels. (G) Effects of fatty acids with varying chain lengths on cell viability under normoxic and hypoxic conditions. (H) Assessment of lipid peroxidation levels under hypoxia using flow cytometry. Statistical analysis involved one-way ANOVA with Tukey's test (A-C) and Student’s t-test (F, G). *P < 0.05, **P < 0.01, ***P < 0.001