Gpr43

(S)-2-(4-chlorophenyl)-3,3-dimethyl-N-(5-phenylthiazol-2-yl) butanamide (CFMB) and TNF-α antagonist R-7050 [44 (link)] (Calbiochem, Darmstadt, Germany); sodium propionate (NaP) (Sigma-Aldrich, St. Louis, MO, USA); pertussis toxin (PTX) (Wako, Osaka, Japan); gallein, cyclopropanecarboxylic acid (CPC) and trichostatin A (TSA) (Tokyo Chemical Industry, Tokyo, Japan); human recombinant TNF-α (PeproTech, Rocky Hill, NJ, USA); polyclonal rabbit antibodies against human β-actin (Abcam), acetyl-histone H3 (Lys9/Lys14), cleaved caspase-3 (Asp175), cleaved caspase-9 (all Cell Signaling Technology, Boston, MA USA), GPR41 (Abcam), and GPR43 (Bioss, Boston, MA, USA); monoclonal rabbit antibodies against HDAC1, HDAC4, HDAC5 (Cell Signaling Technology) and HDAC8 (Abcam); monoclonal mouse antibodies against HDAC3 and caspase-8 (Cell Signaling Technology); and horseradish peroxidase-conjugated anti-mouse, anti-goat and anti-rabbit immunoglobulins (Dako, Glostrup, Denmark) were used in the study.

For immunohistochemical staining, 3 μm paraffin sections were placed in 10 mM sodium citrate buffer (pH=6.0) and heated in a microwave oven or pressure cooker for antigen unmasking after deparaffinization and rehydration. Then, the sections were blocked with 3% hydrogen peroxide and 5% bovine serum albumin. Next, the sections were reacted with primary antibodies against LDLr (1:200 dilution, Abcam, Cambridge, UK), HMGR (1:200 dilution, Bioss, Beijing, China), CD36 (1:400 dilution, Novus, Colorado, USA), CXCL16 (1:100 dilution, Bioss, Beijing, China ) and G protein coupled receptor 43 (GPR43) (1:200 dilution, Bioss, Beijing, China) overnight at 4 °C and subsequently incubated with biotin-labelled secondary antibodies. Finally, the sections were stained with diaminobenzidine and then discontinued in water when a brown colour was detected.
For immunofluorescence staining, cells were fixed and then treated with 0.02% Triton X-100 for 15 min followed by blocking with 5% BSA for 1 h. Next, the cells were incubated with primary antibodies against LDLr, HMGR, CD36, CXCL16 and GPR43 overnight at 4 °C, followed by treatment with secondary antibodies conjugated to Alexa Fluor 555 for 1 h. Nuclei were stained with 4,6-diamidino-2-phenylindole. Finally, cells were examined by Olympus fluorescence microscopy.