Fig 2: Lack of AMPKα2 blocks DHA-induced trans-localization of GPR120 to plasma membrane and interaction with β-arrestin 2.A Representative Quantification of GPR120 mRNA expression in WT and AMPKα2−/− VSMCs (n = 4). B, C Western blotting and quantification of GPR120 expression in primary WT and AMPKα2−/− VSMCs (n = 2). D–F Western blotting and quantification of IL-6 (D) and MCP-1 (E) expression in WT and AMPKα2−/− VSMCs transfected with Ctr-siRNA or Ffar4-siRNA and treated with PA (300 μM) and/or DHA (50 μM). G Representative immunofluorescence images showing GPR120 localization in WT and AMPKα2−/− VSMCs, before and after a 1-h DHA treatment. Scale bar, 25 μm. H–K Representative western blots (H, J) and quantification (I, K) of GPR120 expression in cytoplasmic and membrane fractions before and after a 1-h DHA treatment (n = 4). L, M Western blot and quantification of β-arrestin 2 expression in WT and AMPKα2−/− VSMCs (n = 3). N, O IP assays for studying GPR120–β-arrestin 2 interactions in WT and AMPKα2−/− VSMCs. All data shown represent one of two separate experiments. Quantification of western blots was performed using Image-Pro plus software, distinctly from loading controls. Data are presented as the mean ± s.e.m. p value by two-sided Student’s t tests. Ctr-siRNA control siRNA, Ffar4-siRNA siRNA for mouse Ffar4, IB immunoblot, IP immunoprecipitation.

Fig 3: GPR120 SUMOylation at K32 blocked the effects of DHA in AMPKα2−/− cells.A, B IP assays showed endogenous GPR120 and SUMOylated GPR120 in WT and AMPKα2−/− VSMCs by western blotting of whole cell extracts, prepared in the presence of isopeptidase inhibitors (C, D) IP assays for whole cell extracts from 293T cells co-transfected with either the WT GPR120-tGFP or K32R/GPR120-tGFP vector and the SUMO2-HA vector. E, F Western blotting (E) and quantification (F) of tGFP expression in membrane fractions from AMPKα2−/− VSMCs transfected with GPR120-tGFP or K32R/GPR120-tGFP and treated for 1 h with DHA. Scale bars, 25 μm. G–I Western blotting (G) and quantification of IL-6 (H) and MCP-1 (I) expression in AMPKα2−/− VSMCs transfected with GPR120-tGFP or K32R/GPR120-tGFP and treated with PA (300 μM) and DHA (50 μM) for 1 h. All IP and western blotting data shown represent one of two separate experiments, distinctly from loading controls. Quantification of western blot results was performed using Image-Pro plus software. Data are presented as the mean ± s.e.m. p value by two-sided Student’s t tests. K32R lysine 32 mutated to arginine, PA palmitic acid, DHA docosahexaenoic acid.

Fig 4: Increases in the expression of UBC9 and SUMO2/3 promotes the GPR120 SUMOylation in AMPKα2−/− VSMCs by activating the C-MYC S67 phosphorylation.A, B Western blotting and quantification of UBC9 and GPR120 expression in cytoplasmic and membrane fractions from AMPKα2−/− VSMCs transfected with Ctr-siRNA or Ube2i-siRNA, with or without DHA (50 μM) treatment (n = 4). p value versus Ctr-siRNA by one-way ANOVA tests. C–E Western blotting and quantification of UBC9, IL-6, and MCP-1 expression in AMPKα2−/− VSMCs transfected with Ctr-siRNA or Ube2i-siRNA before or after PA (300 μM) and DHA (50 μM) treatment for 1 h (n = 2). F, G Western blotting and quantification of c-myc, UBC9, and SUMO2/3 expression in AMPKα2−/− VSMCs transfected with Ctr-siRNA or Myc-siRNA (n = 4). H–K Western blotting (H) and quantification of tGFP (I), UBC9 (J), and SUMO2/3 (K) expression in 293T cells transfected with WT C-myc (WT), S64A/C-myc (S64A), or S67A/C-myc (S67A) vectors and treated for 2 hrs with AICAR (1 mM) (n = 2). L Extracts from 293T cells transfected with either WT, S64A, or S67A c-myc constructs were prepared to assess the interactions between the c-myc-tGFP fusion protein and AMPKα2 or pAMPKα. M Recombinant AMPK and recombinanted c-myc-tGFP protein were incubated in the kinase buffer with ATP. Immunoprecipitation was done using anti-tGFP antibody. N The phosphorylation of serine at amino acids 67 in the peptide PTPPLSPSRRSG by LC-MS/MS followed processed using Proteome Discoverer 1.3. O Representative images of immunostaining showing AMPKα2 and UBC9 expression and localization in human artery tissues from control (n = 4) and CAD patients (n = 8). Scale bar, 400 μM. P Semi-quantification analysis of immunostaining using Image-Pro plus software. Correlational analyses of AMPKα2 expression and the expression of UBC9 (Q) in human artery tissues. All data shown represent one of three separate experiments. Quantification of western blots was performed using Image-Pro plus software, distinctly from loading controls. Data are presented as the mean ± s.e.m. p value by two-sided Student’s t tests. AICAR 5-aminoimidazole-4-carboxamide 1-α-d-ribofuranoside, Ube2i-siRNA siRNA against mouse Ube2i, Myc-siRNA siRNA against mouse Myc, CAD coronary artery disease.

Fig 5: AMPKα2 was required for the anti-inflammatory effects of FO in vivo.Serum MCP-1 (A) and IL-6 (B) levels were measured in LDLR−/− and LDLR−/−/AMPKα2−/− mice fed a western diet with or without 5% FO treatment (n = 6). Representative western blot (C) and quantification of MCP-1 (D) and IL-6 (E) and protein levels in aortic tissues from LDLR−/− and LDLR−/−/AMPKα2−/− mice fed a western diet for 12 weeks, with or without 5% FO administration (n = 3), distinctly from loading controls. ELISAs for MCP-1 (F) and IL-6 (G) in aortic tissue lysates from LDLR−/− and LDLR−/−/AMPKα2−/− mice with or without 5% FO administration (n = 3). H Representative immunofluorescence images for SM α-actin and CD68 in aortic tissues of LDLR−/− and LDLR−/−/AMPKα2−/− mice, with or without 5% FO administration. I, J Quantification of CD68 area and ratio in aortic tissues of LDLR−/− and LDLR−/−/AMPKα2−/− mice, with or without 5% FO administration (n = 4). Scale bars, 200 μm. The data shown represent one of two separate experiments. All values are expressed as means ± s.e.m. p value vs LDLR−/− mice by two-sided Student’s t tests. FO fish oils, MCP-1 monocyte chemoattractant protein-1, IL-6 interlukin-6.