Fig 2: Screening several cancers cells lines to identify the potential target(s) of compounds with antiproliferative activities using competitive ABPP. (A) Western blot analysis of the expression of endogenous MAGL, ABHD6, and ABHD12 in the cancer cell lines. (B) Comparative ABPP analysis of the potential targets labeled by serine hydrolase probe FP-TAMRA. (C–J) Competitive ABPP gel results of compounds 1, 9, 17, 23, 24, 64, 82, 93, 95, 98, 101, 102, 104, 127, and 154 show the selectivity profile across serine hydrolases labeled by FP-TAMRA (500 nM). Note that the concentration of KML29, KT182, and DO264 was 10 µM, respectively. (K) Heatmap overview of the number of potential targets (identified by the quantification of protein bands in ABPP) for compounds with antiproliferative activities in various cancer cells.

Fig 3: Fluorescence turn-on responses of AA-HNA on 2-AG hydrolases (MAGL, ABHD6, and ABHD12). (A) Proposed mechanism of AA-HNA catalyzed by 2-AG hydrolases (MAGL, ABHD6, and ABHD12). (B) Absorbance spectra of AA-HNA and HNA. (C) Fluorescence spectra response of AA-HNA after incubation (30 min) with MAGL, ABHD6, or ABHD12. (D) Concentration-dependent fluorescence spectra response at the emission of 455 nm with the increasing of MAGL concentration (0–100 ng/µl) in the presence of AA-HNA (10 µM). (E) Titration curve of AA-HNA (10 µM) with increasing concentration of MAGL (0–100 ng/µl), and the linear relationship was observed in the range of 0–25 ng/µl.

Fig 4: Semaglutide directly acts on hepatic GLP-1R to regulate miR-5120/ABHD6 expression, and affects ABHD6 expression through miR-5120 in vitro. (A) The expression of miR-5120 in Hepa 1–6 cells stimulated by HG+FFA+Sema plus GLP-1R siRNA was detected (n = 6). (B) The mRNA expression of Abhd6 in Hepa 1–6 cells stimulated by HG+FFA+Sema plus GLP-1R siRNA or miR-5120 inhibitors was detected (n = 6). (C) ABHD6 protein expression in Hepa 1–6 cells stimulated by HG+FFA+Sema plus GLP-1R siRNA or miR-5120 inhibitors was measured by Western blotting. (D) The mRNA expression of Glp-1r in hepatocytes was measured (n = 6). (E) The protein expression of GLP-1R in hepatocytes was measured by immunofluorescence staining (400×). (F) The mRNA expression of Glp-1r in liver tissues (n = 6). (G) The protein expression of GLP-1R in liver tissues was measured. (H) The protein expression of GLP-1R in Hepa 1–6 cells after transfection with three GLP-1R SiRNAs. Data are expressed as mean ± SD, *P < 0.05, **P < 0.01 vs Control group; #P < 0.05, ##P < 0.01 vs T2DM+NAFLD group or HG+FFA group; &P < 0.05, &&P < 0.01 vs HG+FFA+Sema group.

Fig 5: Semaglutide decreases ABHD6 and increases miR-5120 expression in the liver of T2DM combined with NAFLD mice and in hepatocytes stimulated by HG+FFA. (A) The mRNA expression of Abhd6 in liver tissues (n = 6). (B) The protein expression of ABHD6 in liver tissues. (C) The mRNA expression of Abhd6 in hepatocytes (n = 6). (D) The protein expression of ABHD6 in hepatocytes. (E) The expression of hepatic and serum miR-5120 was detected (n = 6). (F) miR-5120 mRNA levels of Hepa 1–6 cells and supernatant were measured (n = 6). (G) The expression of miR-5120 in Hepa 1–6 cells activated by miR-5120 mimics was measured by qPCR (n = 6). (H) The mRNA expression of Abhd6 in Hepa 1–6 cells stimulated by miR-5120 mimics was detected (n = 6). (I) ABHD6 protein level in Hepa 1–6 cells stimulated by miR-5120 mimics was determined. (J) The expression of miR-5120 in Hepa 1–6 cells suppressed by miR-1a inhibitors was measured (n = 6). (K) The mRNA expression of Abhd6 in Hepa 1–6 cells stimulated by miR-5120 inhibitors was detected (n = 6). (L) ABHD6 protein level in Hepa 1–6 cells stimulated by miR-1a inhibitors was determined. (M) Dual-Luciferase reporter assay verified that ABHD6 was a target gene of miR-5120 (n = 3). Data are expressed as mean ± SD, *P < 0.05, **P < 0.01 vs control group; #P < 0.05, ##P < 0.01 vs T2DM+NAFLD group.