Guang-Rong Yan

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