differentiation 36  (R&D Systems)

Journal: Frontiers in Immunology

Article Title: Discovery and in vitro characterization of a human anti-CD36 scFv

doi: 10.3389/fimmu.2025.1531171

Figure Lengend Snippet: Identification of the scFv anti-CD36 D11 (A) Strategy of panning of the ALTHEA Gold Libraries™ for identification of anti-CD36 scFvs. (B) Phage ELISA for 90 randomly selected clones from the third round of panning. Graph shows the signals generated by binding to rhCD36 (red) or protein L (ProL; brown). As control, we detected rhCD36 with the commercial antibody JC63.1 (blue). (C) Determination of structural integrity of purified scFv D11 by denaturing SDS-PAGE. R, reducing condition; NR, non-reducing condition; MW, molecular weight. (D, E) ELISA assay evaluating the binding of purified D11 (green) to protein L (D) or rhCD36 (E) . JC63.1 (blue) was used as a positive control and specificity was assessed by using the unrelated scFv P5E1A6 (black). (F) Representative competition ELISA (from two performed) evaluating the binding to rhCD36 captured by the reference antibody JC63.1 in presence of D11 (green) or the specificity control P5E1A6 (black). The signal generated in the absence of D11 is shown in red. (G) Number of clones identified at each stage of the discovery process. (G) was created with Biorender.com .

Article Snippet: Nunc Maxisorb plates (Thermo Scientific, Cat. 278743) were coated with 50 µg/mL recombinant human CD36 (rhCD36) from Sino Biological (Cat. 10752-H08H) in PBS overnight at 4°C.

Techniques: Enzyme-linked Immunosorbent Assay, Clone Assay, Generated, Binding Assay, Control, Purification, SDS Page, Molecular Weight, Positive Control

Journal: Frontiers in Immunology

Article Title: Discovery and in vitro characterization of a human anti-CD36 scFv

doi: 10.3389/fimmu.2025.1531171

Figure Lengend Snippet: Binding of D11 to membrane CD36. (A) CD36 expression in macrophage-like THP-1 cells, as detected with the anti-CD36 FA6-152 (pink). The fluorescence generated by staining with the secondary antibody alone is shown in gray. (B) Concentration-response curve evaluating the binding of D11 to CD36 + macrophage-like THP-1 cells. Data from two independent experiments. Representative histograms are shown on the right. (C) CD36 expression in HepG2 cells. (D) Binding of D11 (green) to HepG2 cells and representative histograms (right). Data are representative of two independent experiments, each one including duplicates. (B, D) show the signals from the secondary antibody anti-His-tag PE (gray) and the unrelated scFv P5E1A6 (black).

Article Snippet: Nunc Maxisorb plates (Thermo Scientific, Cat. 278743) were coated with 50 µg/mL recombinant human CD36 (rhCD36) from Sino Biological (Cat. 10752-H08H) in PBS overnight at 4°C.

Techniques: Binding Assay, Membrane, Expressing, Fluorescence, Generated, Staining, Concentration Assay

Journal: Frontiers in Immunology

Article Title: Discovery and in vitro characterization of a human anti-CD36 scFv

doi: 10.3389/fimmu.2025.1531171

Figure Lengend Snippet: Effect of D11 in the uptake of CD36 ligands. (A) Analysis of oxLDL-DyLigth uptake in macrophage-like THP-1 cells. Cells were treated with D11 (green), JC63.1 (blue), or the unrelated P5E1A6 (black). Response was normalized against the MFI of untreated cells. Data from three independent experiments. (B) Representative histograms of the effect of the different treatments (100 µg/mL) on the uptake of oxLDL. (C) Effect of the listed antibodies (100 µg/mL) on the uptake of BODIPY-palmitate in HepG2 cells. Bars in the graph show the percentage of cells with BODIPY high fluorescence from three independent experiments (mean ± SEM). *p < 0.05; **p < 0.01, ns: not significant, Bonferroni’s multiple comparisons test. (D) Representative dot plots of the experiments reported in (C) .

Article Snippet: Nunc Maxisorb plates (Thermo Scientific, Cat. 278743) were coated with 50 µg/mL recombinant human CD36 (rhCD36) from Sino Biological (Cat. 10752-H08H) in PBS overnight at 4°C.

Techniques: Fluorescence

Journal: Frontiers in Immunology

Article Title: Discovery and in vitro characterization of a human anti-CD36 scFv

doi: 10.3389/fimmu.2025.1531171

Figure Lengend Snippet: Effect of D11 on the oxLDL-induced foam cell phenotype. (A) Effect of treatments on LD accumulation in macrophage-like THP-1 cells exposed to oxLDL (50 μg/ml) for 16-18 h. Graph shows the quantification (mean ± SEM) of oil red staining from two independent experiments. **p < 0.01; ****p < 0.0001, Bonferroni´s multiple comparisons test. (B) Representative micrographs of the experiments reported in (A) . (C-E) Effect of treatments on the relative expression of CD36 (C) , SRA1 (D) , and ACAT (E) . Graphs show the geometric mean ± geometric SD from three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001, ns: not significant, Bonferroni´s multiple comparisons test.

Article Snippet: Nunc Maxisorb plates (Thermo Scientific, Cat. 278743) were coated with 50 µg/mL recombinant human CD36 (rhCD36) from Sino Biological (Cat. 10752-H08H) in PBS overnight at 4°C.

Techniques: Staining, Expressing

Journal: Frontiers in Immunology

Article Title: Discovery and in vitro characterization of a human anti-CD36 scFv

doi: 10.3389/fimmu.2025.1531171

Figure Lengend Snippet: D11 impairs LD accumulation and clonogenicity on HepG2 cells. (A) Experimental protocol for the analysis of phenotypic changes induced by CD36 blockage. (B) Effect of treatments on LD accumulation. The bars in the graph (mean ± SEM) show the oil red-stained area from two independent experiments. **p < 0.01; ***p < 0.001; ****p < 0.0001, ns: not significant, Bonferroni´s multiple comparisons test. (C) Representative micrographs of the results presented in (B) . (D) Effect of treatments (100 µg/mL) on CD36 mRNA expression. Graph shows the geometric mean ± geometric SD from two independent experiments. *p < 0.05; **p < 0.01, ns: not significant, Bonferroni´s multiple comparisons test. (E) Experimental protocol for the evaluation of the effect of scFv D11 on the clonogenicity of HepG2 cells. (F) Effect of treatments on sphere formation efficiency (SFE). Data (mean ± SEM) from three independent experiments. *p < 0.05; ****p < 0.0001, ns: not significant, Bonferroni´s multiple comparisons test. (G) Representative micrographs of the results presented in (F) , with insets showing enlarged images of tumorspheres (scale=100 µm). (A, E) were created with Biorender.com .

Article Snippet: Nunc Maxisorb plates (Thermo Scientific, Cat. 278743) were coated with 50 µg/mL recombinant human CD36 (rhCD36) from Sino Biological (Cat. 10752-H08H) in PBS overnight at 4°C.

Techniques: Staining, Expressing

Journal: Biomolecules

Article Title: Melanoma-Derived Extracellular Vesicles Induce CD36-Mediated Pre-Metastatic Niche

doi: 10.3390/biom14070837

Figure Lengend Snippet: Role of CD36 in the clinical prognosis and immune infiltration correlation in cutaneous melanoma: ( A ) Hallmark enrichment plot of CD36 generated with core cancer hallmark gene set ( n = 1574) shows that CD36 is associated with invasion and metastasis of melanoma as well as reprogramming energy metabolism ( p < 0.05). ( B ) Progression-free survival with CD36 expression in melanoma patients, using upper (red) and lower (blue) quartile data from the SKCM dataset. ( C ) Correlation of CD36 with CD163, CD14, CD209, and FAP in SKCM dataset ( n = 471). ( D ) CD36 positively correlates with the infiltration level of M2 macrophages in the SKCM-metastasis dataset ( n = 368). ( E ) Correlation between CD36 and infiltration level of endothelial cells in SKCM-metastasis dataset ( n = 368).

Article Snippet: Recombinant Human CD36 Protein was loaded for CD36 positive control (catalog: 10752-H08H, Sino Biological, Chesterbrook, PA, USA).

Techniques: Generated, Expressing

Journal: Biomolecules

Article Title: Melanoma-Derived Extracellular Vesicles Induce CD36-Mediated Pre-Metastatic Niche

doi: 10.3390/biom14070837

Figure Lengend Snippet: Melanoma-derived EVs upregulate CD36 expression in the recipient cells. ( A ) Brief procedure for LEV and melanoma-cell-derived EV collection and characterization. Summarily, afflicted afferent lymph channels were surgically excised, and effluents of channels were utilized to collect LEVs through size-exclusion chromatography. Cell line culture-conditioned media was used to collect EVs by ultracentrifugation. All collected EV characterizations were carried out with NTA (see method in for more details). ( B ) LEVs collected from patients possess a significantly higher percentage of CD36 + EVs in total LEVs than control LEVs. ( C ) SKMEL28 and C32TG melanoma-cell-derived EVs increase expression of CD36 in HLEC (endothelial) and THP1 (monocytic) cells after 24 h of exposures. ( D ) Microscopic observation of CFSE-labeled-SKMEL28-derived EVs in HLEC cells (scale bar: 100 µm). ( E ) ImageStream analysis of CFSE-labeled-SKMEL28-derived EVs in THP1 cells (scale bar: 7 µm). ( F ) The box plot indicates the real-time PCR data by Cq values of CD36 and GAPDH gene expression in EVs collected from melanoma cell lines. Original images of ( A , C ) can be found in .

Article Snippet: Recombinant Human CD36 Protein was loaded for CD36 positive control (catalog: 10752-H08H, Sino Biological, Chesterbrook, PA, USA).

Techniques: Derivative Assay, Expressing, Size-exclusion Chromatography, Control, Labeling, Real-time Polymerase Chain Reaction

Journal: Biomolecules

Article Title: Melanoma-Derived Extracellular Vesicles Induce CD36-Mediated Pre-Metastatic Niche

doi: 10.3390/biom14070837

Figure Lengend Snippet: Melanoma-derived EVs regulate M2-macrophage-like characteristics by upregulating CD36. ( A ) CD36 surface expression was increased upon EV exposure to cells compared to the control in M0 macrophages derived from THP1 cells. ( B ) Melanoma-derived EV exposure increases M2-macrophage-like characteristics by upregulating CD163 in PMA-pretreated THP1 cells. ( C ) EVs collected from CD36-silenced SKMEL28 cells decrease M2 macrophage characteristics in THP1-treated cells through CD36 downregulation.

Article Snippet: Recombinant Human CD36 Protein was loaded for CD36 positive control (catalog: 10752-H08H, Sino Biological, Chesterbrook, PA, USA).

Techniques: Derivative Assay, Expressing, Control

Journal: Biomolecules

Article Title: Melanoma-Derived Extracellular Vesicles Induce CD36-Mediated Pre-Metastatic Niche

doi: 10.3390/biom14070837

Figure Lengend Snippet: Spatial MxIF image analysis of SLN (−), SLN (+) tissue from patients, and LN tissue from control subjects. ( A ) Box plot showing the percentage of cells expressing a specific marker of all cells in a field of view (FOV) (center panel) and proportion of CD36+ cells per FOV in the three groups of samples. ( B ) Estimated probability of CD36 cells colocalized with other immune cells (within a 40-micron radius of a CD36 cell) in an analyzed panel of markers using spatial regression models.

Article Snippet: Recombinant Human CD36 Protein was loaded for CD36 positive control (catalog: 10752-H08H, Sino Biological, Chesterbrook, PA, USA).

Techniques: Control, Expressing, Marker

Journal: Biomolecules

Article Title: Melanoma-Derived Extracellular Vesicles Induce CD36-Mediated Pre-Metastatic Niche

doi: 10.3390/biom14070837

Figure Lengend Snippet: CD36 cell colocalization spatial regression model results of melanoma SLN (−) compared to control LN.

Article Snippet: Recombinant Human CD36 Protein was loaded for CD36 positive control (catalog: 10752-H08H, Sino Biological, Chesterbrook, PA, USA).

Techniques: Control

Journal: Biomolecules

Article Title: Melanoma-Derived Extracellular Vesicles Induce CD36-Mediated Pre-Metastatic Niche

doi: 10.3390/biom14070837

Figure Lengend Snippet: Primary-melanoma-secreted EVs in lymphatic fluid enter the SLN via afferent lymphatic vessels. The SLN components, including macrophages and endothelial cells, receive these EVs to upregulate CD36, possibly through CD36 cargoes (mRNA and protein). The upregulation of CD36 in the targeted cells can mediate the immunosuppression mechanism to promote the tumor-promoting niche in the SLN.

Article Snippet: Recombinant Human CD36 Protein was loaded for CD36 positive control (catalog: 10752-H08H, Sino Biological, Chesterbrook, PA, USA).

Techniques:

Journal: Theranostics

Article Title: Fatty acid-induced CD36 expression via O-GlcNAcylation drives gastric cancer metastasis

doi: 10.7150/thno.34024

Figure Lengend Snippet: PA treatment promoted metastasis and induced CD36 expression through activating the HBP. (A) GFAT and OGT 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 3 h. The values shown are expressed as the means ± SD of three independent experiments. (B) GFAT, OGT and CD36 levels in MKN-45 or SGC 7901 cells were assessed by western blotting after treatment with the indicated concentration of PA or control solvent for 3 h. (C) The levels of O-GlcNAcylation in MKN-45 or SGC 7901 cells were assessed by western blotting after treatment with 10 μM TMG or isometric DMSO for 24 h. β-actin was used as a loading control. (D) Transwell migration and invasion assay of MKN-45 and SGC 7901 cells after10 μM TMG or isometric DMSO treatment for 24 h. (E) Indicated cells were injected into nude mice (n = 10 for each group) via the tail vein and mice were fed with HFD. Animals were sacrificed at 6 weeks after the injections. Representative HE staining of the metastatic nodes in mouse lungs from each group. (F) 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. * represents Mann Whitney test p < 0.05.

Article Snippet: In vitro O-GlcNAcylation assay: In vitro O-GlcNAcylation of CD36-FL (ORIGENE, TP710013, Rockville, USA) or CD36-ES (Sino Biological, 10752-H08H, Beijing, China) was performed in 100 μL assay volumes containing 1 μg of OGT (R&D, 8446-GT, Minneapolis, USA) in reaction buffer (50 mM Tris-HCl, 1 mM DTT, and 12.5 mM MgCl 2 , pH 7.5), and 2 mM UDP-GlcNAc (Calbiochem®, 670107, San Diego, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Concentration Assay, Western Blot, Migration, Invasion Assay, Injection, Staining, MANN-WHITNEY

Journal: Theranostics

Article Title: Fatty acid-induced CD36 expression via O-GlcNAcylation drives gastric cancer metastasis

doi: 10.7150/thno.34024

Figure Lengend Snippet: 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.

Article Snippet: In vitro O-GlcNAcylation assay: In vitro O-GlcNAcylation of CD36-FL (ORIGENE, TP710013, Rockville, USA) or CD36-ES (Sino Biological, 10752-H08H, Beijing, China) was performed in 100 μL assay volumes containing 1 μg of OGT (R&D, 8446-GT, Minneapolis, USA) in reaction buffer (50 mM Tris-HCl, 1 mM DTT, and 12.5 mM MgCl 2 , pH 7.5), and 2 mM UDP-GlcNAc (Calbiochem®, 670107, San Diego, USA).

Techniques: Real-time Polymerase Chain Reaction, Knock-Out, Concentration Assay, Luciferase, Reporter Assay, Activity Assay, Transduction, Western Blot

Journal: Theranostics

Article Title: Fatty acid-induced CD36 expression via O-GlcNAcylation drives gastric cancer metastasis

doi: 10.7150/thno.34024

Figure Lengend Snippet: 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.

Article Snippet: In vitro O-GlcNAcylation assay: In vitro O-GlcNAcylation of CD36-FL (ORIGENE, TP710013, Rockville, USA) or CD36-ES (Sino Biological, 10752-H08H, Beijing, China) was performed in 100 μL assay volumes containing 1 μg of OGT (R&D, 8446-GT, Minneapolis, USA) in reaction buffer (50 mM Tris-HCl, 1 mM DTT, and 12.5 mM MgCl 2 , pH 7.5), and 2 mM UDP-GlcNAc (Calbiochem®, 670107, San Diego, USA).

Techniques: Modification, Activity Assay, Expressing, Western Blot, Recombinant, Transfection, Lysis, Staining, Fluorescence, Plasmid Preparation, Transwell Assay, Injection, MANN-WHITNEY

Journal: Theranostics

Article Title: Fatty acid-induced CD36 expression via O-GlcNAcylation drives gastric cancer metastasis

doi: 10.7150/thno.34024

Figure Lengend Snippet: 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.

Article Snippet: In vitro O-GlcNAcylation assay: In vitro O-GlcNAcylation of CD36-FL (ORIGENE, TP710013, Rockville, USA) or CD36-ES (Sino Biological, 10752-H08H, Beijing, China) was performed in 100 μL assay volumes containing 1 μg of OGT (R&D, 8446-GT, Minneapolis, USA) in reaction buffer (50 mM Tris-HCl, 1 mM DTT, and 12.5 mM MgCl 2 , pH 7.5), and 2 mM UDP-GlcNAc (Calbiochem®, 670107, San Diego, USA).

Techniques: Expressing