11,(12)-epoxy-5 z ,8 z ,14 z -eicosatrienoic acid (11,12-eet  (Cayman Chemical)

Journal: Nature Communications

Article Title: Androgen receptor promotes arachidonic acid metabolism and angiogenic microenvironment in AFP-negative hepatocellular carcinoma

doi: 10.1038/s41467-025-61448-z

Figure Lengend Snippet: a Differentially expressed genes (DEGs) calculated from scRNA-seq data between AFP − HCC and AFP + HCC. b Pathways enriched in tumor cells in AFP – HCC and AFP + HCC from scRNA-seq data by GSEA. The p -values, calculated by permutation test, of all listed pathways were less than 0.05. c The volcano plot from scRNA-seq data manifested differentially metabolic activity of tumor cells in AFP – HCC when compared to AFP + HCC. The color and dot size correspond to the log 2 FC in metabolic activity between AFP − HCC and AFP + HCC. d Correlation analysis between metabolic pathways and transcriptomic AFP expression in scRNA-seq data by Spearman correlation test. e Metabolic pathway diagram of arachidonic acid metabolism and gene signature scores of three branches in arachidonic acid metabolism. Box plot depicts the median and interquartile range, and the lower and upper hinges denote the 25–75% interquartile range (IQR), with whiskers extending up to a maximum of 1.5 times IQR. f Scatter plot showing negative correlation between transcriptomic AFP expression and CYP gene signature in scRNA-seq data by Spearman correlation test. g The heatmap of genes involved in arachidonic acid metabolism in AFP – HCC and AFP + HCC, colored by average expression value. h Relative expression of CYP2C8/CYP1B1 detected by RT-qPCR in tumor tissues from 15 AFP − HCC and 15 AFP + HCC patients. Data were shown as mean ± SD. i The volcano plot shows metabolites detected by targeted lipidomics. The abbreviations ARA and DTA stand for arachidonic acid and docosatetraenoic acid, respectively. j The expression levels of AFP were detected by RT-qPCR and western blot in HCC cell lines. k ELISA analysis revealed elevated levels of 11,12-EET in the CM derived from AFP low cell lines compared to AFP high cell lines. Data were shown as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 by Wilcoxon rank sum test in ( e, g , i ), two-sided t-test in ( h ) and one-way ANOVA with Tukey’s multiple comparisons test ( j , k ). Source data are provided as a Source Data file.

Article Snippet: The concentration of 11,12-EET in both the CM and the tissue was measured using the enzyme-linked immunosorbent assay (ELISA) kit (11E39-K01, Eagle Biosciences), according to the manufacturer’s instructions.

Techniques: Activity Assay, Expressing, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Derivative Assay

Journal: Nature Communications

Article Title: Androgen receptor promotes arachidonic acid metabolism and angiogenic microenvironment in AFP-negative hepatocellular carcinoma

doi: 10.1038/s41467-025-61448-z

Figure Lengend Snippet: a SCENIC analysis in scRNA-seq data shows TF activities in AFP − HCC and AFP + HCC tumor cells. b Spearman correlation analysis between activity of TFs listed in ( a ), and AFP expression (colored in blue) as well as CYP genes signature (colored in red) in tumor cells. c Detection of AR expression using IHC in AFP – HCC ( n = 10) and AFP + HCC ( n = 10). Scale bars, 50 μm. d Correlation between AFP and AR expression by Spearman correlation test ( n = 20). e Relative expression of AR detected by RT-qPCR (AFP − HCC vs. AFP + HCC = 15:15). f AR expression in AFP – HCC compared to AFP + HCC, as determined by bulk RNA-seq data from FAH-SYSU (AFP – HCC vs. AFP + HCC = 161:273) and TCGA-LIHC cohort (AFP – HCC vs. AFP + HCC = 142:127). g Concentrations of testosterone and 5α-DHT detected by lipidomics using blood plasma from 10 AFP − /AFP + HCC. h Relative expression of AR detected by RT-qPCR and western blot in HCC cells. i Relative concentration of 11,12-EET detected by ELISA assays in CM from HCC cells. j Relative expression of CYP2C8 and CYP1B1 with or without 5α-DHT stimulation. k Validation of the AR knockdown (shAR) in MHCC97H and HCCLM3 using western blot. l 11,12-EET concentrations detected by ELISA assays in CM from MHCC97H and HCCLM3 cells (shNC) compared to their shAR counterparts. m Detection of 11,12-EET levels in AR-overexpressing Hepa1-6 cells (oeAr) versus control cells (oeNC). Validation of AR binding with CYP2C8 and CYP1B1 promoters by ChIP-PCR ( n ) and ChIP-qPCR ( o ) in MHCC97H and HCCLM3. p Normalized snATAC-seq tracks of AFP , AR , CYP2C8 and CYP1B1 . Three biological replicates were employed ( h – j, l , m , o ). Data were shown as mean ± SD ( c, g – j, l, m , o ). * p < 0.05, ** p < 0.01, *** p < 0.001 and ns stands for no significance by Wilcoxon rank sum test ( f , g ), two-sided t-test in ( c, e, i , j , m ) and one-way ANOVA with Tukey’s multiple comparisons test ( h , l , o ). Source data are provided as a Source Data file.

Article Snippet: The concentration of 11,12-EET in both the CM and the tissue was measured using the enzyme-linked immunosorbent assay (ELISA) kit (11E39-K01, Eagle Biosciences), according to the manufacturer’s instructions.

Techniques: Activity Assay, Expressing, Quantitative RT-PCR, RNA Sequencing, Clinical Proteomics, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay, Biomarker Discovery, Knockdown, Control, Binding Assay, ChIP-qPCR

Journal: Nature Communications

Article Title: Androgen receptor promotes arachidonic acid metabolism and angiogenic microenvironment in AFP-negative hepatocellular carcinoma

doi: 10.1038/s41467-025-61448-z

Figure Lengend Snippet: a Experimental design using NCG mice ( n = 6 per group). Representative images of tumors ( b ), tumor growth curve ( c ), tumor volume measurements ( d ) and tumor weight measurements ( e ) in MHCC97H subcutaneous HCC mouse models (shNC vs. shAR = 6:6). Representative images of tumors ( f ), tumor growth curve ( g ), tumor volume measurements ( h ) and tumor weight measurements ( i ) in Huh7 subcutaneous HCC mouse models (oeNC vs. oeAR = 6:6). j Relative concentrations of 11,12-EET measured by ELISA assays in MHCC97H and Huh7 subcutaneous HCC tumors, and their corresponding shAR and oeAR counterparts ( n = 6 per group), respectively. Flow cytometry analysis of CD45 - CD31 + EGFR + endothelial cell (EC) fractions in MHCC97H ( k ) and Huh7 ( l ) subcutaneous HCC tumors ( n = 6 per group). m Experimental design using C57BL/6 mice ( n = 6 per group). Representative images of tumors ( n ), tumor growth curve ( o ), tumor volume measurements ( p ) and tumor weight measurements ( q ) in RIL-175 subcutaneous HCC mouse models (shNC vs. shAr = 6:6). Representative images of tumors ( r ), tumor growth curve ( s ), tumor volume measurements ( t ) and tumor weight measurements ( u ) from Hepa1-6 subcutaneous HCC mouse models (oeNC vs. oeAr = 6:6). v Relative concentration of 11,12-EET detected by ELISA kit in RIL-175 and Hepa1-6 subcutaneous HCC tumors, and their corresponding shAr and oeAr counterpart ( n = 6 per group), respectively. Flow cytometry analysis of EGFR + ECs fractions in RIL-175 ( w ) and Hepa1-6 ( x ) subcutaneous HCC tumors ( n = 6 per group). Data were shown as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 by two-way ANOVA in ( c , g , o , s ) and two-sided t-test in ( d , e , h – l , p – q , t – x ). Source data are provided as a Source Data file.

Article Snippet: The concentration of 11,12-EET in both the CM and the tissue was measured using the enzyme-linked immunosorbent assay (ELISA) kit (11E39-K01, Eagle Biosciences), according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Flow Cytometry, Concentration Assay