Fig 2: Sarcolemmal CD36 interacted with Src kinase to initiate PA-induced Src/Akt/ERK1/2 kinase activation in LUAD cells.A PA promoted co-localization between sarcolemmal CD36 and Src. NCI-H23 and A549 cells were treated with or without 500 μM PA for 0.5 h respectively. Cells were double stained for Src (red) and CD36 (green) respectively. Bar, 5 μm. The images were representative of three experiments. Linear scan the fluorescence intensity was done to determine the distribution of Src and CD36 by scanning the cell cross section along the straight line with Zeiss microscope software. B, C PA promoted the interaction between CD36 and Src. NCI-H23 cells were treated with or without 500 μM PA for 0.5 h. Total cell lysis (input) was detected with indicated antibodies. CD36 was immunoprecipitated by its own antibody and then the immunoprecipitates were examined with Src antibodies (Ctr: non-PA treatment). Similarly, Src immunoprecipitates (IP by Src antibody) were examined with CD36 antibody. D CD36 initiated PA-induced Src/Akt/ERK1/2 signaling pathway. NCI-H23 and A549 cells were treated with or without 500 μM PA for 0.5 h, or cells were pretreated with 50 μM SSO (a fatty acid analog as CD36 blocker) and 10 μM Dasatinib (Src inhibitor) for 15 min respectively, followed by 500 μM PA treatment for another 30 min. Then proteins were examined with indicated antibodies.

Fig 3: CD36 knockdown in LUAD cells inhibited xenograft tumor growth, aggressiveness and metastasis.A CD36 knockdown in LUAD cells inhibited xenograft tumor growth. The nude mice fed with NCD and HFD were subcutaneously transplanted with A549-Scramble and A549-ShCD36 cells respectively. The tumor was collected 5 weeks later and the size of tumor was measured (n = 10, *p < 0.05 and **p < 0.01 vs. Scramble NCD; ##p < 0.01 vs. Scramble HFD). B Measurement of plasma free fatty acids (FFA), total cholesterol (TC) and triglyceride (TG). FFA, TC and TG in mice blood were measured with kits according to the instructions. **p < 0.01 vs. Scramble NCD, ##p < 0.01 vs. ShCD36 HFD, n = 5. C CD36 knockdown in LUAD cells inhibited xenograft tumor aggressiveness. Subcutaneous tumor sections from A549-Scramble NCD, A549-Scramble HFD, A549-ShCD36 NCD, or A549-ShCD36 HFD were stained with H&E and then examined. The poorly differentiated and more aggressive cells in A549-Scramble HFD tumors (Spindle cells (red arrows), cells with high nuclear-cytoplasm ratio (green arrows), mitotic cells (black arrows) were indicated. D CD36 knockdown in LUAD cells inhibited actin-remodeling in subcutaneous tumor cells of HFD-fed mice. Subcutaneous tumor sections were stained for actin (red). The representative IF staining image of tumors generated from the mice of A549-Scramble NCD, A549-Scramble HFD, A549-ShCD36 NCD, or A549-ShCD36 HFD were shown. Bar, 5 μm. The images were representative of three experiments. E CD36 knockdown in LUAD cells inhibited colocalization between Arp2 and actin in subcutaneous tumor cells of HFD-fed mice. Subcutaneous tumor sections were double stained for actin (red) and Arp2 (green) respectively. The representative IF staining image of tumors generated from the mice of A549-Scramble NCD, A549-Scramble HFD, A549-ShCD36 NCD, or A549-ShCD36 HFD were shown. Bar, 5 μm. The images were representative of three experiments. F Detection of lung metastasis of subcutaneous tumor cells. After subcutaneous tumor removal, the mice were continued to be fed with NCD or HFD for another 12 weeks and then sacrificed. Lungs of nude mice were collected and LUAD cell metastasis to lung were examined and lung metastatic nodules were calculated. Lung surface nodules were indicated as black arrows. Data were mean ± SD, *p < 0.05 vs. Scramble NCD, #p < 0.05 vs. Scramble HFD, n = 4. G Pathological analysis of the lungs from subcutaneous tumor removal mice. Lung tissue sections from mice of A549-Scramble NCD, A549-Scramble HFD, A549-ShCD36 NCD, or A549-ShCD36 HFD were stained with H&E and examined. H IHC staining of the lungs from subcutaneous tumor removal mice. Lung tissue sections from mice of A549-Scramble NCD, A549-Scramble HFD, A549-ShCD36 NCD, or A549-ShCD36 HFD were stained with CD36 and examined.

Fig 4: CD36-Src signal was required for PA-induced actin remodeling.A, B CD36 distribution and actin remodeling upon PA stimulation. NCI-H23 and A549 cells were treated with or without 500 μM PA for 0.5 h, respectively. Then cells were double stained for actin (red) and CD36 (green) respectively. Bar, 5 μm. The images were representative of three experiments. C PA upregulated the activation of Src/Akt/ERK1/2 via CD36. Cells were treated with or without 500 μM PA for 0.5 h. Untreated cells were set up as controls. Then proteins were examined with corresponding antibodies as indicated. Ctr: non-PA treatment. D, E CD36 was required for PA-induced actin remodeling. Scramble and ShCD36 LUAD cells were treated with or without 500 μM PA for 0.5 h. Cells were stained for actin (red). Cells given green fluorescence contained the GFP protein, proving the stable transfected cells as indicated in the Materials and Methods. Bar, 5 μm. The images were representative of three experiments. F CD36-Src signal was required for PA-induced actin remodeling. NCI-H23 and A549 cells were treated with or without 500 μM PA for 0.5 h, or cells were pretreated with 50 μM SSO (a fatty acid analog as CD36 blocker), 100 nM SU6656 (Src inhibitor) and 10 μM Dasatinib (Src inhibitor) respectively for 15 min, followed by 500 μM PA treatment for another 0.5 h. Then cells were double stained for actin (red) and CD36 (green) respectively. Bar, 5 μm. The images were representative of three experiments.

Fig 5: CD36 translocated to plasma membrane of LUAD cell upon PA stimulation.A CD36 distribution on the cell membrane was enhanced by PA treatment. NCI-H23 and A549 cells were treated with or without 500 μM PA for 0.5 h respectively. Cells were double stained for CD36 (green) and Na-K ATPase (red) respectively. Bar, 5 μm. The images were representative of three experiments. Linear scan the fluorescence intensity was done to determine the CD36 distribution by scanning the cell cross section along the straight line with Zeiss microscope software. B PA increased the content of CD36 on plasma membrane. NCI-H23 and A549 cells were treated with or without 500 μM PA for 30 min. Subcellular fractionation was performed to purify the plasma and cytosol fractions. Equal amount proteins were subjected to immunoblot for CD36 with corresponding antibodies. The images were representative of three experiments (Ctr: non-PA treatment).