Fig 1: Macrophage-specific CD147 knockout inhibits foam cell formation. (A) Generation of macrophage-specific CD147 knockout mice (Lyz2cre/+CD147f/f, namely, CD147M–KO mice) is illustrated by the mating scheme. The control WT littermates were Lyz2+/+CD147f/f, namely, CD147WT mice. PCR analysis of genomic DNA showed the genotyping. The fragments from top to bottom are the KI Cre gene, WT Cre gene, and floxed and WT CD147 gene. Genomic DNA from Lyz2cre/+ mice was used as the P.C. for Cre analysis. Genomic DNA from CD147f/+ mice was used as the P.C. for CD147 analysis. H2O was used as the N.C. (B) Characterization of BMDMs isolated from CD147WT or CD147M–KO mice via western blotting and RT-PCR. (C) Representative images of Oil Red O staining of CD147WT or CD147M–KO BMDMs that were incubated with or without ox-LDL (50 µg/mL) for 24 h. The scale bar is 50 µm. (D) For quantification, Oil Red O absorbance was measured at 492 nm after extraction with isopropanol. (E) BMDMs from CD147WT or CD147M–KO mice stimulated with or without ox-LDL (50 µg/mL) for 24 h were stained with Bodipy (lipids, green), F4/80 (macrophages, red), and DAPI (nuclei, blue) and examined via confocal microscopy. The scale bar is 20 µm. (F) The total cholesterol and cholesteryl ester contents were determined with a coupled enzyme assay. (G) Representative images of Filipin staining of BMDMs from CD147WT or CD147M–KO mice treated with or without ox-LDL (50 µg/mL) for 24 h. The scale bar is 100 µm. Data represent the mean ± SEM. of n = 3 biologically independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Fig 2: Effect of FUT1 transfection on the expression of MMP-2, and the effect of anti-Lewis y antibody and anti-CD147 antibody on the expression of MMP-2. Western blot detection of the expression of MMP-2 in RMG-I and RMG-I-hFUT cells, and in the absence and presence of anti-Lewis y antibody and anti-CD147 antibody, respectively. (A) Representative western blots of MMP-2 in the cell lines. (B) Densitometric quantification of the protein expression (n=3). For the inhibition assay, the final concentration of anti-Lewis y antibody was 20 µg/ml and the final concentration of anti-CD147 antibody was 10 µg/ml. The duration of treatment was 1 h. *P<0.05, vs. RMG-I; **P<0.05, vs. RMG-I or RMG-I-hFUT cells without anti-Lewis y antibody or anti-CD147 antibody treatment. FUT1, a1,2-fucosyltransferase; MMP-2, matrix metalloproteinase; mAb, monoclonal antibody; N, RMG-I cells; T, RMG-I-hFUT cells.
Fig 3: Compound screening identifies PI3K/Akt/mTOR signaling as a regulator of CD147 expression induced by ox-LDL in macrophages. (A) WT BMDMs treated with or without ox-LDL (50 µg/mL) for 24 h were analyzed by RNA sequencing. Identification of DEGs is illustrated in a volcano plot. (B) KEGG pathway enrichment histogram showing genes involved in foam cell formation based on the upregulated DEGs. (C) Overview of a large compound screening by RT-PCR showing CD147 mRNA levels in BMDMs incubated for 1 h with one of 46 potential compounds targeting 26 candidate signaling pathways and then treated with ox-LDL (50 µg/mL) for 24 h. (D) RT-PCR analysis of CD147 mRNA levels in BMDMs treated with ox-LDL (50 µg/mL) for 24 h in the presence or absence of 3-MA (1 mM), wortmannin (100 nM), MK-2206 2HCl (1 µM), or Rapamycin (100 nM). (E–H) Western blot analysis of phosphorylated (p-) and total PI3K, Akt, and mTOR and CD147 in BMDMs exposed to 50 µg/mL ox-LDL for 24 h in the presence or absence of 3-MA (1 mM), wortmannin (100 nM), MK-2206 2HCl (1 µM), or Rapamycin (100 nM). Data represent the mean ± SEM. of n = 3 biologically independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Fig 4: Macrophage-specific CD147 deficiency diminishes CD36 expression and may exert other protective effects in atherosclerosis. (A) BMDMs from CD147WT and CD147M–KO treated with or without ox-LDL (50 µg/mL) for 24 h were analyzed by RNA sequencing. The selected genes involved in foam cell formation were graphed in a heatmap. (B) Western blot analysis of LDLR, ABCA1, ABCG1, SR-A, and CD36 in BMDMs isolated from CD147M–KO and CD147M–KI mice and their respective CD147WT littermates incubated with or without ox-LDL (50 µg/mL) for 24 h. (C) Quantification of protein levels of molecules in (B) relative to tubulin. (D) Immunofluorescence staining of CD147, CD36, ABCA1, ABCG1, and LDLR in BMDMs isolated from CD147M–KO and CD147M–KI mice and their respective CD147WT littermates treated with ox-LDL (50 µg/mL) for 24 h. Scale bar, 20 µm. (E) Quantification of the MFI of (D) by means of the Image-Pro Plus 6.0 software. (F,G) GESA GO enrichment analysis for biological process (F) and GESA reactome enrichment analysis (G) are shown as lollipop charts. Statistical significance in gene sets was defined as an FDR < 0.25; NES represents enrichment magnitude and is shown as the size of the point. The -log10 (FDR) of the enrichment is shown on the x-axis; the enriched gene set is shown on the y-axis; and the color of points represent the fraction of gene sets that are significantly upregulated or downregulated. Data represent the mean ± SEM. of n = 3 biologically independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Fig 5: Inhibition of CD147 attenuates early proinflammatory activation of Ly-6C+ monocytes/macrophages (MMs) in the spleen at 4 h tMCAO. a Representative western blot images showing protein levels of CCR2 and Ly-6C detected in isolated splenocytes from the following groups (n = 5 mice/group): sham, tMCAO, tMCAO + isotype, and tMCAO + aCD147. Similar results were obtained in three independent experiments. No difference was noted between tMCAO and +iso groups. b RT-qPCR analysis of mRNA levels of CCR2 and Ly-6C detected in isolated splenocytes from the following groups (n = 5 mice/group): sham surgery, tMCAO + isotype, and tMCAO+aCD147. *p < 0.05 vs. sham control; #p < 0.05 vs. tMCAO + isotype. c Representative dot plots illustrating the flow cytometry gating strategy for identification of immune cell subsets in the spleen. Monocyte subsets in the spleen were identified by selecting the gate (R1) APC-CD11bhigh and PE-Linlow and further separated by CD11c/F4/80-PeCy5 and Ly-6C-FITC. d Flow cytometric quantification of Ly-6Chigh and Ly-6Clow monocyte subsets. The number and ratio of Ly-6Chigh and Ly-6Clow monocyte subsets in spleen were measured in the following groups (n = 5 mice/group): sham surgery, tMCAO + isotype, and tMCAO + aCD147. *p < 0.05 vs. sham; #p < 0.05 vs. tMCAO + isotype. e RT-qPCR analysis of mRNA levels of iNOS, IL-6, and Arg1 detected in the FACS-sorted splenic monocytes from the indicated groups of mice (n = 6/groups). *p < 0.05 vs. sham control; #p < 0.05 vs. tMCAO + isotype
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