Journal: bioRxiv

Article Title: CD36-Pyruvate Kinase M2 Signaling Promotes Macrophage Phagocytosis Through Mitochondrial Reactive Oxygen Species

doi: 10.1101/2023.09.07.556574

Figure Lengend Snippet: A , a diagram of the ex vivo mtROS and phagocytosis assay. B , Total amount of aortic F4/80 + /Trem2 + macrophages were quantified and shown in the bar graph; n=4-7 individual mice per group. C , MitoNeoD MFI was quantified in aortic F4/80 + /Trem2 + macrophages and shown in the bar graph; n=5 individual mice per group. D , pHrodo MFI was quantified in aortic F4/80 + /Trem2 + macrophages and shown in the bar graph; n=5-6 individual mice per group. E , a diagram of the in vivo phagocytosis assay. F , pHrodo MFI was quantified in aortic F4/80 + /Trem2 + macrophages and shown in the bar graph; n=5-7 individual mice per group.

Article Snippet: For phagocytosis assays, the filtered single-cell suspension was incubated with 20 μg/ml pHrodo-conjugated E. coli bioparticles in RPMI1640 with 10% FBS at 37°C for 30 min, followed by immunostaining with PE/Cy5-conjugated F4/80 antibody (BioLegend, Cat#123111) and PE-conjugated Trem2 antibody (R&D Systems, Cat#FAB17291P) in Flow Buffer at RT for 15 min in the dark.

Techniques: Ex Vivo, Phagocytosis Assay, In Vivo

Journal: bioRxiv

Article Title: CD36-Pyruvate Kinase M2 Signaling Promotes Macrophage Phagocytosis Through Mitochondrial Reactive Oxygen Species

doi: 10.1101/2023.09.07.556574

Figure Lengend Snippet: A-D , Mouse scRNA-seq data were re-analyzed from a previous publication . Uniform manifold approximation and projection (UMAP) representation of 11 aortic CD45 + immune cell clusters were shown in A . Trem2 gene expression pattern ( B ) and Pkm gene expression pattern ( C ) were shown in the UMAP. D , Violin plots show the Pkm and Trem2 expression distribution among aortic macrophage subpopulations. ResMac: resident macrophages; InflaMac: inflammatory macrophages. E , HMDMs transfected with PKM siRNA were treated with 50 μg/ml oxLDL for 24 h before subjected to mtROS assay (left panel) or phagocytosis assay (right panel). MFI was quantified and shown in the bar graph; n=4-5 per group. F , Representative confocal images of macrophages immunostained for PKM2 (green) and Tom20 (red). Nuclei were stained by DAPI (blue). Scale bar: 5 μm. G , HMDMs treated with 20 μg/ml LDL (control) or oxLDL for 3 h were lysed, subjected to cell fractionation into mitochondrial and cytosol fractions. PKM2 and ATP5A (mitochondria fraction loading control) blot images from mitochondrial fractions were shown on the left. PKM2 and β-actin (cytosol fraction loading control) blot images from cytosol fractions were shown on the right. Images were quantified, normalized to each loading control, and expressed as fold change of control. n=4 per group. H , WT or Cd36 -null peritoneal macrophages treated with 20 μg/ml LDL (control) or oxLDL for 3 h and then processed as in G. Mitochondrial fractions were immunoblotted for PKM2 and ATP5A and blot images were shown. Images were quantified, normalized, and expressed as fold change of control. n=4 per group. I , WT macrophages treated with 20 μg/ml oxLDL or pre-treated 1 or 5μM shikonin before addition of oxLDL, incubating for 3 h, and then processed as in G. Mitochondrial fractions were immunoblotted for PKM2 and Tom20 and blot images were shown. Images were quantified and expressed as fold change of control. n=3 per group. J , WT macrophages pre-treated with 20 μg/ml oxLDL or in combination with 1 μM shikonin for 24 h before mtROS or phagocytosis assay. The MitoNeoD (left) or pHrodo (right) MFI was quantified and shown in the bar graph; n=3-4 per group.

Article Snippet: For phagocytosis assays, the filtered single-cell suspension was incubated with 20 μg/ml pHrodo-conjugated E. coli bioparticles in RPMI1640 with 10% FBS at 37°C for 30 min, followed by immunostaining with PE/Cy5-conjugated F4/80 antibody (BioLegend, Cat#123111) and PE-conjugated Trem2 antibody (R&D Systems, Cat#FAB17291P) in Flow Buffer at RT for 15 min in the dark.

Techniques: Gene Expression, Expressing, Transfection, Phagocytosis Assay, Staining, Control, Cell Fractionation