Fig 2: 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 3: O-GlcNAcylation modification of CD36 in S468 and T470 enhances its FA uptake activity. (A) The O-GlcNAc sites of CD36 predicted by the YinOYang 1.2 server (www.cbs.dtu.dk/services/YinOYang) are shown with a black arrowhead at the top. The green vertical lines show the potential O-GlcNAc-modified Ser/Thr residues, and the red horizontal wavy line indicates the threshold for modification potential. (B) Total lysates from SGC 7901 cells expressing FLAG-tagged CD36 (FLAG-CD36) were subjected to IP with FLAG Ab, followed by western blotting using the indicated antibodies (Abs). (C) Total lysates from SGC 7901 cells expressing FLAG-tagged CD36 (FLAG-CD36) with or without TMG were subjected to IP with IgG or FLAG, followed by western blotting using O-GlcNAcylation or FLAG antibodies. (D) Schematic model of the recombinant CD36 full length (CD36-FL) protein and CD36 extracellular segment (CD36-ES). (E) O-GlcNAcylation assays using recombinant human CD36-FL or CD36-ES were performed in the presence or absence of UDP-GlcNAc or recombinant human OGT. Reaction mixtures were immunoblotted with antibodies against O-GlcNAc (RL2), CD36, and OGT. (F, G) Lentiviral vectors encoding FLAG-tagged wild-type (WT) CD36, CD36 with an alanine substitution at S468 (S468A), CD36 with an alanine substitution at T470 (T470A) or CD36 with alanine substitutions at both S468 and T470 (S468A/T470A) were transfected into SGC 7901 cells after knocking out endogenous CD36 expression. Then, the cells were treated with 10 µM TMG or isometric DMSO for 12 h before cell lysis, and then the O-GlcNAcylation of CD36 was detected. (H) Representative Nile red staining of SGC 7901 cells transfected with WT, S468A, T470A or S468A &T470A CD36 lentiviral vectors after treatment with 0.3 µM of PA or isometric control solvent for 24 h. (I) Fluorescence of SGC 7901 cells transfected with the indicated lentiviral vector. Fluorescence was measured at the indicated times after the addition of uptake reaction mix. Data are expressed as the mean RFU ± SD of three independent plates, with each data point representing the mean of quadruplicate wells. * represents Student's t test p < 0.05. (J) Transwell assay of SGC 7901 cells transfected with the indicated lentiviral vector after 0.4 µM PA treatment for 24 h. (K) Indicated SGC 7901 cells were injected into the tail vein of nude mice (n = 6 for each group) fed either a HFD or a normal chow diet. Photos of representative lung tissue samples in each group are shown. 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.

Fig 4: 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.

Fig 5: Upregulation of CD36 predicts poor survival in GC. (A) CD36 gene copy number in 305 GC tumor samples and 94 normal controls from TCGA data. The data were analyzed by ONCOMINE. (B,C) Kaplan-Meier curve depicting the OS of 876 GC patients (B) and the progression-free survival of 641 GC patients (C). The data were analyzed by a Kaplan-Meier plotter (http://kmplot.com/analysis/). (D) Representative images of IHC staining of CD36 in primary tumor tissues and positive metastatic lymph nodes of three GC patients.