Journal: Cell Death & Disease

Article Title: The activated CD36-Src axis promotes lung adenocarcinoma cell proliferation and actin remodeling-involved metastasis in high-fat environment

doi: 10.1038/s41419-023-06078-3

Figure Lengend Snippet: A CD36 knockdown and B CD36 overexpression LUAD cell lines construction. Lentivirus transfection was performed to construct cell lines of NCI-H23-Scramble, NCI-H23-ShCD36, A549-Scramble, A549-ShCD36, NCI-H23-Vector, NCI-H23-CD36OE-FLAG, A549-Vector, and A549-CD36OE-FLAG. The efficiency of knockdown or overexpression was confirmed by CD36 protein examination. C , D Cell proliferation assay. Cells were treated with PA for 6 h, then BrdU assay was performed. 0 μM PA (non-treatment) 6 h was set up as 1, * p < 0.05 and ** p < 0.01 vs. scramble, # p < 0.05 and ## p < 0.01 vs . scramble+PA, n = 6. $ p < 0.05 and $$ p < 0.01 vs. vector, ^ p < 0 .05 and ^^ p < 0.01 vs. vector+PA , n = 6. E , F Cell migration and G , H invasion assay . Cells were treated with 10 μM PA for 24 h were applied for trans-well assay to detect cell migration and invasion respectively. Images were taken using light microscopy (scale bar: 50 μm). The numbers of migration or invasion cells in seven randomly selected fields were counted and the average number of cells in one field was calculated and expressed as the mean ± SD. * p < 0.05 and ** p < 0.01 vs scramble/vector respectively; # p < 0.05 and ## p < 0.01 vs scramble/vector+PA respectively, n = 3.

Article Snippet: WB antibodies against CD36 (ab133625) were purchased from Abcam (Cambridge, MA), antibodies against Src (A19119), Rac1 (A5539), Cdc42 (A1188) and RhoA (A15641) were from Abclonal (Wuhan, China), antibodies against p-Src-Tyr416 (6943S), p-Src-Tyr527 (2105S), p-Akt473 (9271S), Akt (9272S), p-ERK1/2 (4370S), ERK1/2 (4695S), MMP-9 (13667S), GAPDH (5174S), anti-mouse (7076) and anti-rabbit (7074) HRP-linked IgGs, were purchased from Cell Signaling (Boston, MA).

Techniques: Over Expression, Transfection, Construct, Plasmid Preparation, Proliferation Assay, BrdU Staining, Migration, Invasion Assay, Light Microscopy

Journal: Cell Death & Disease

Article Title: The activated CD36-Src axis promotes lung adenocarcinoma cell proliferation and actin remodeling-involved metastasis in high-fat environment

doi: 10.1038/s41419-023-06078-3

Figure Lengend Snippet: 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).

Article Snippet: WB antibodies against CD36 (ab133625) were purchased from Abcam (Cambridge, MA), antibodies against Src (A19119), Rac1 (A5539), Cdc42 (A1188) and RhoA (A15641) were from Abclonal (Wuhan, China), antibodies against p-Src-Tyr416 (6943S), p-Src-Tyr527 (2105S), p-Akt473 (9271S), Akt (9272S), p-ERK1/2 (4370S), ERK1/2 (4695S), MMP-9 (13667S), GAPDH (5174S), anti-mouse (7076) and anti-rabbit (7074) HRP-linked IgGs, were purchased from Cell Signaling (Boston, MA).

Techniques: Staining, Fluorescence, Microscopy, Software, Fractionation, Western Blot

Journal: Cell Death & Disease

Article Title: The activated CD36-Src axis promotes lung adenocarcinoma cell proliferation and actin remodeling-involved metastasis in high-fat environment

doi: 10.1038/s41419-023-06078-3

Figure Lengend Snippet: 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.

Article Snippet: WB antibodies against CD36 (ab133625) were purchased from Abcam (Cambridge, MA), antibodies against Src (A19119), Rac1 (A5539), Cdc42 (A1188) and RhoA (A15641) were from Abclonal (Wuhan, China), antibodies against p-Src-Tyr416 (6943S), p-Src-Tyr527 (2105S), p-Akt473 (9271S), Akt (9272S), p-ERK1/2 (4370S), ERK1/2 (4695S), MMP-9 (13667S), GAPDH (5174S), anti-mouse (7076) and anti-rabbit (7074) HRP-linked IgGs, were purchased from Cell Signaling (Boston, MA).

Techniques: Staining, Fluorescence, Microscopy, Software, Lysis, Immunoprecipitation

Journal: Cell Death & Disease

Article Title: The activated CD36-Src axis promotes lung adenocarcinoma cell proliferation and actin remodeling-involved metastasis in high-fat environment

doi: 10.1038/s41419-023-06078-3

Figure Lengend Snippet: 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.

Article Snippet: WB antibodies against CD36 (ab133625) were purchased from Abcam (Cambridge, MA), antibodies against Src (A19119), Rac1 (A5539), Cdc42 (A1188) and RhoA (A15641) were from Abclonal (Wuhan, China), antibodies against p-Src-Tyr416 (6943S), p-Src-Tyr527 (2105S), p-Akt473 (9271S), Akt (9272S), p-ERK1/2 (4370S), ERK1/2 (4695S), MMP-9 (13667S), GAPDH (5174S), anti-mouse (7076) and anti-rabbit (7074) HRP-linked IgGs, were purchased from Cell Signaling (Boston, MA).

Techniques: Staining, Activation Assay, Fluorescence, Transfection

Journal: Cell Death & Disease

Article Title: The activated CD36-Src axis promotes lung adenocarcinoma cell proliferation and actin remodeling-involved metastasis in high-fat environment

doi: 10.1038/s41419-023-06078-3

Figure Lengend Snippet: A PA activated Rac1 in a CD36-Src signal-dependent manner. 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 10μM Dasatinib (Src inhibitor) for 15 min, respectively, which was followed by 500 μM PA treatment for another 0.5 h. The GTP binding Rac1, Cdc42, and RhoA were detected with kits as mentioned in Materials and Methods and their expression was examined with antibodies as indicated. B Akt kinase but not ERK regulated Rac1 activity. NCI-H23 and A549 cells were treated with or without 500 μM PA for 0.5 h, or cells were pretreated with MEK/ERK inhibitor U0126 (10 μM), and Akt inhibitor API2 (20 μM) for 15 min respectively, which was followed by 500 μM PA treatment for another 0.5 h. GTP binding Rac1 and its expression were detected. C Rac1 colocalized with the finger like actin remodeling after 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 actin (red) and Rac1 (green) respectively. Bar, 5 μm. The images were representative of three experiments. D Rac1 was required for PA-induced actin remodeling. NCI-H23 and A549 cells were transiently transfected with the HA-tagged wild type Rac1 (Rac1 WT), dominant negative Rac1 (Rac1 DN) and constitutively active Rac1 (Rac1 CA) respectively. After 48 h of transfection, cells were treated with or without 500 μM PA for 0.5 h. Then cells were double stained for actin (red) and HA (green) respectively. Bar, 5 μm. The images were representative of three experiments.

Article Snippet: WB antibodies against CD36 (ab133625) were purchased from Abcam (Cambridge, MA), antibodies against Src (A19119), Rac1 (A5539), Cdc42 (A1188) and RhoA (A15641) were from Abclonal (Wuhan, China), antibodies against p-Src-Tyr416 (6943S), p-Src-Tyr527 (2105S), p-Akt473 (9271S), Akt (9272S), p-ERK1/2 (4370S), ERK1/2 (4695S), MMP-9 (13667S), GAPDH (5174S), anti-mouse (7076) and anti-rabbit (7074) HRP-linked IgGs, were purchased from Cell Signaling (Boston, MA).

Techniques: Binding Assay, Expressing, Activity Assay, Staining, Transfection, Dominant Negative Mutation

Journal: Cell Death & Disease

Article Title: The activated CD36-Src axis promotes lung adenocarcinoma cell proliferation and actin remodeling-involved metastasis in high-fat environment

doi: 10.1038/s41419-023-06078-3

Figure Lengend Snippet: 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.

Article Snippet: WB antibodies against CD36 (ab133625) were purchased from Abcam (Cambridge, MA), antibodies against Src (A19119), Rac1 (A5539), Cdc42 (A1188) and RhoA (A15641) were from Abclonal (Wuhan, China), antibodies against p-Src-Tyr416 (6943S), p-Src-Tyr527 (2105S), p-Akt473 (9271S), Akt (9272S), p-ERK1/2 (4370S), ERK1/2 (4695S), MMP-9 (13667S), GAPDH (5174S), anti-mouse (7076) and anti-rabbit (7074) HRP-linked IgGs, were purchased from Cell Signaling (Boston, MA).

Techniques: Staining, Generated, Immunohistochemistry

Journal: Cell Death & Disease

Article Title: The activated CD36-Src axis promotes lung adenocarcinoma cell proliferation and actin remodeling-involved metastasis in high-fat environment

doi: 10.1038/s41419-023-06078-3

Figure Lengend Snippet: A Detection of lung metastasis of tail-vein injected LUAD cells. The nude mice fed with NCD and HFD were injected with A549-Scramble or A549-ShCD36 cells through tail-vein. After 12 weeks, the lungs of nude mice were collected 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. B Pathologic analyses of the lungs from tail-vein injected LUAD cell 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. C IHC staining of the lungs from tail-vein injected LUAD cell 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. D SSO inhibited PA-enhanced LUAD cell viability. A549 cells were treated with or without 10 μM PA for 48 h, or cells were pretreated with 50 μM SSO for 30 min, which was followed by 10 μM PA treatment or not for another 48 h. MTT assay were performed and non-treatment condition was set up as 1, ** p < 0.01 vs. control, ## p < 0.01 vs. PA + SSO, n = 5. E SSO inhibited PA-induced LUAD cell proliferation. A549 cells were treated with or without 10 μM PA for 6 h, or cells were pretreated with 50 μM SSO for 30 min, which was followed by 10 μM PA treatment for another 6 h. BrdU assay were performed and non-treatment 6 h condition was set up as 1, ** p < 0.01 vs. control, ## p < 0.01 vs. PA + SSO, n = 6. F SSO treatment inhibited LUAD cell metastasis in vivo. A549 cells were treated with or without 50 μM SSO for 24 h. Then the cells were injected into nude mice fed with NCD and HFD respectively through tail-vein. The mice were killed after 12 weeks. The lung metastatic nodules were calculated. Data were mean ± SD, ** p < 0.01 vs. NCD, # p < 0.05 vs. HFD, n = 5. G Schematic mechanism illustrating the high fat-induced LUAD cells metastasis. HFD leads to increased levels of fatty acids in the body. Upon binding with fatty acid, the CD36 molecules located on plasma membrane function as the initiator of CD36-Src-Akt/ERK signaling, which in turn activates Rac1 via Akt, and up-regulates MMP-9 expression via Akt and ERK. All of these contribute to the fatty acid-induced redistribution of Arp2/3, MMP-9 and cortactin, leading to actin remodeling-involved LUAD cell pulmonary metastasis in high-fat environment.

Article Snippet: WB antibodies against CD36 (ab133625) were purchased from Abcam (Cambridge, MA), antibodies against Src (A19119), Rac1 (A5539), Cdc42 (A1188) and RhoA (A15641) were from Abclonal (Wuhan, China), antibodies against p-Src-Tyr416 (6943S), p-Src-Tyr527 (2105S), p-Akt473 (9271S), Akt (9272S), p-ERK1/2 (4370S), ERK1/2 (4695S), MMP-9 (13667S), GAPDH (5174S), anti-mouse (7076) and anti-rabbit (7074) HRP-linked IgGs, were purchased from Cell Signaling (Boston, MA).

Techniques: Injection, Staining, Immunohistochemistry, MTT Assay, BrdU Staining, In Vivo, Binding Assay, Expressing