Fig 2: Upregulation of CD36 in GC cell promotes cell metastasis. (A) Flow cytometry analysis showing the expression of CD36 in SGC 7901-NM cells transfected with a lentiviral vector encoding CD36 or negative control vector. K isotype IgG was used as a negative control. (B)3D spheroid BME cell invasion assay of SGC 7901-NM cells transfected with lentiviral vector encoding CD36 (LV-CD36) or with negative control vector (LV-NC). Photographs of all the spheroids in each well every 24 h for 7 days using a 4× objective. Quantitative analysis of the surface area of all spheroids. Normalized areas for all the spheroids are presented relative to the area on the first day. All of the areas were calculated three times using ImageJ, and the values represent the means ± SD. (C) The indicated cells were injected into nude mice (n = 6 for each group) via the tail vein along with weekly intraperitoneal injections of 20 µg of the anti-CD36 neutralizing monoclonal antibody JC63.1 or 20 µg of the corresponding IgA. Animals were sacrificed at 8 weeks after the injections. Photos of representative lung tissue samples in each group are shown. (D) Left: The histogram shows the proportion of mice with lung metastasis in each group. “Met” is short for “metastasis,” and “Met-free” indicates “metastasis-free.” Right: Mann Whitney test was used to evaluate the number of metastatic nodes in the lungs of mice from each group.

Fig 3: Association of CD36 and OGT expression with the prognosis of GC patients. (A-C) Kaplan-Meier curve depicting the OS and progression-free survival of a publicly available cohort of 300 GC patients (GSE62254). High and low expression of CD36 or OGT were defined by patients whose tumors expressed CD36 or OGT at levels higher and lower than the median, respectively. (D-G) Area under the receiver-operating characteristic (AUROC) curves of CD36 or OGT or both were used to diagnose the indicated patients in all 300 GC patients from GSE62254. (H) Schematic model of Fatty acid-induced CD36 expression via O-GlcNAcylation drives gastric cancer metastasis.

Fig 4: O-GlcNAcylation promoted CD36 transcription via activating the NF-κB pathway. (A) CD36 mRNA levels in MKN-45 or SGC 7901 cells were assessed by real-time PCR after a 24-hour treatment with TMG (10 μM) or isometric DMSO. (B) CD36 mRNA levels were assessed in MKN-45 or SGC 7901 OGT-knockout cells by real-time PCR. (C) CD36 mRNA levels in SGC 7901 cells with or without OGT knockout after treatment with the indicated concentration of PA for 24 h. (D) A luciferase reporter assay showed changes in the activity of 45 signal transduction pathways in SGC 7901 cells after a 24-hour treatment with TMG (10 μM). (E) A luciferase reporter assay showed the regulation of CD36 transcription by the indicated transcription factors in HEK 293T cells. (F) The levels of O-GlcNAcylation, RELA, phosphorylated RELA and CD36 in MKN-45 or SGC 7901 cells were assessed by western blotting after treatment with 10 μM TMG or isometric DMSO for the indicated times. β-actin was used as a loading control. (G) A luciferase reporter assay showed the regulation of CD36 transcription by NF-κB after treatment with 10 μM TMG or isometric DMSO for 12 h. (H) CD36 mRNA levels in MKN-45 or SGC 7901 cells were assessed by real-time PCR after the indicated treatment. The values shown are expressed as the means ± SD of three independent experiments. Control: control solvent treatment for 24 h; PA: treatment with 0.4 μM of PA for 24 h; PA+PDTC: treatment with 0.4 μM of PA for 24 h and pretreatment with 50 µmol of pyrrolidine dithiocarbamate for 4 h; DMSO: isometric DMSO treatment for 24 h; TMG: treatment with 10 μM of TMG for 24 h; TMG+PDTC: treatment with 10 μM of TMG for 24 h and pretreatment with 50 µmol of pyrrolidine dithiocarbamate for 4 h.

Fig 5: High fat diets induced CD36 expression and promoted metastasis in mice. (A) SGC 7901 cells were injected into the tail vein of nude mice (n = 9 for each group) fed either a HFD or a normal chow diet. Photos of representative lung tissue samples in each group are shown. (B) Left: The histogram shows the proportion of mice with lung metastasis in each group. “Met” is short for “metastasis,” and “Met-free” represents “metastasis-free.” Right: Mann Whitney test was used to evaluate the number of metastatic nodes in the lungs of each group. (C) Representative CD36 staining of the metastatic nodes in mouse lungs from the HFD or normal chow diet groups. (D) CD36 mRNA levels in MKN-45 or SGC 7901 cells were assessed by real-time PCR after treatment with the indicated concentration of PA or control solvent for 24 h. The values shown are expressed as the means ± SD of three independent experiments. (E) Flow cytometry analysis of CD36 levels in MKN-45 or SGC 7901 cells after treatment with the indicated concentration of PA or control solvent for 24 h. K isotype IgG was used as a negative control. (F, G) Immunofluorescence staining showed the FA and CD36 levels in SGC 7901 cells without (F) or with (G) 0.4 µM PA treatment for 24 h. FAs were stained by Nile red.