Ceramide c6

Amyloid-β1–42 (Aβ1–42) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Before injection, the Aβ1–42 peptide was dissolved in a physiological saline solution at a concentration of 5 mg/ml and incubated at 37°C for 72 h to induce aggregation. Human full length BDNF (ab9794), NGF (ab138794) and NT-3 (ab138798) proteins and their corresponding rabbit polyclonal antibodies (ab75040, ab6198 and ab65804) were purchased from Abcam (Cambridge, UK). Rabbit monoclonal antibodies against phospho-ERK (#4370), total ERK (#4695), phospho-JNK (#4668), total JNK (#9258), phospho-p38 (#4511), total p38 (#9212) and GAPDH (#5174) were from Cell Signaling Technology (Beverly, MA, USA). Ceramide C6, an ERK activator, was obtained from Santa Cruz Technology (Santa Cruz, CA, USA). The ERK inhibitor PD98059 was from Sigma-Aldrich (St. Louis, MO, USA).

Eggs with embryos were incubated at 37 °C in an incubator with 70% humidity. After 6 to 7 days of incubation, an air chamber was made, the shell membrane and chorioallantoic membrane were separated, and a window was opened on the shell membrane for injection. The CM from GC-shESM1 cells with or without recombinant ESM1, MAPK/ERK inhibitor SCH772984 (Selleck, Houston, TX, USA) or c-Met-inhibitor AMG-337 (Shanghai Ulva test), and the CM from GC cells with or without the ESM1-neutralizing antibody and MAPK/ERK stimulator Ceramide C6 (Santa Cruz, CA, USA) or MAPK/ERK inhibitor SCH772984 (Selleck, USA) were added to the loop by window injection. After 3 days, the CAM area was isolated, unfolded on a glass slide, and imaged under a stereomicroscope. Vascularization was assessed using image J/FIJI.

To clarify the role of ERK signaling in acetate-regulated lipid metabolism induced, the 3-day differentiated cells were treated with 10 μM Ceramide C6 (an ERK1/2 activator, Santa Cruz Technology, Santa Cruz, CA, USA) or vehicle (dimethyl sulfoxide) for 12 h [22 (link)]; the cells were then given a treatment of either acetate (9 μM) or saline for 48 h before collected.

The normal colonic epithelial cell line NCM460 and the CRC cell lines including SW480, HCT116, DLD1, SW1116, Caco-2, SW620 and HT-29 were purchased from ATCC and cultured with DMEM culture medium (Gibco, USA) containing 10% fetal bovine serum (FBS, Gibco, USA), 10 μg/mL streptomycin and 100 U/mL penicillin at 37°C in a 5% CO₂ incubator (Thermo Fisher, USA). For cell transfection, cells in the logarithmic phase were detached with trypsin and seeded in six-well plates at a density of 1×10⁵ cells per well. After 24 h culture, when the cell confluence reached about 75%, the transfection was performed using a FuGENE6 transfection kit (Promega, USA) following the standard instructions provided. Following another 48 h of culture, the transfected cells were harvested and the ANGPTL4 expression was quantified using RT-qPCR. Activation of the ERK pathway was performed by treatment with 10 µM of Ceramide C6 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) 13 (link). The cells were divided into shCtr group (transfected with silencing negative control plasmid), shANGPTL4 group (transfected with shANGPTL4 plasmid), control group (transfected with negative control overexpression plasmid), ANGPTL4 group (transfected with oe-ANGPTL4 plasmid), and ANGPTL4 + Ceramide C6 group (transfected with oe-ANGPTL4 plasmid + Ceramide C6).

Treatment reagents included MG132 (Merk, Schwalbach, Germany), CA (Cell Signaling Technologies, Beverly, MA), OA (Merk), Ceramide C6 (Santa Cruz Biotechnology Inc., Santa Cruz, CA), Cyclohexamide (Merk) and S3I-201 (Merk).