Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Gastric cancer-derived exosomal let-7 g-5p mediated by SERPINE1 promotes macrophage M2 polarization and gastric cancer progression

doi: 10.1186/s13046-024-03269-4

Figure Lengend Snippet: High SERPINE1 expression in GC cells promotes macrophage M2 polarization. tSNE visualization of nine single-cell clusters partitioned by unsupervised cluster analysis, SERPINE1 expression of each single-cell, and SERPINE1 expression abundance of different single-cell clusters in the GSE134520 ( A – C ) and GSE167297 ( D – F ) datasets. ( G ) Flow cytometry analysis of the proportion of CD68 + CD206 + macrophages in a Transwell co-culture system, with MKN45 and AGS cells overexpressing (oe_ SERPINE1 ) or silencing SERPINE1 (shRNA#3 or sh_ SERPINE1 #3) in the upper chamber, and THP1 cells treated with PMA in the lower chamber. ( H ) Immunofluorescence staining of xenograft tumor tissues. Comparison of the proportion of M1 or M2 macrophage infiltration. Green indicates F4/80. Red indicates iNOS or Arg1 expression

Article Snippet: THP-1 cells were differentiated into macrophages using 150 ng/mL phorbol 12-myristate 13-acetate (PMA, Sigma) for 24 h and subsequently co-cultured with cancer-derived exosomes or GC cells in 6-well plates with 0.4-μm membranes for 72 h. Harvested macrophages were converted into single-cell suspensions, stained with Elab Fluor 488 anti-human CD68 (Mouse, 1:20, ElabScience) and APC anti-human CD206 (Mouse, 1:20, ElabScience) antibodies, and analyzed for CD68 + CD206 + populations by flow cytometry (Accuri C6, BD).

Techniques: Expressing, Flow Cytometry, Co-Culture Assay, shRNA, Immunofluorescence, Staining, Comparison

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Gastric cancer-derived exosomal let-7 g-5p mediated by SERPINE1 promotes macrophage M2 polarization and gastric cancer progression

doi: 10.1186/s13046-024-03269-4

Figure Lengend Snippet: SERPINE1 -mediated gastric cancer-derived exosomes facilitate the polarization of THP1 cells into M2 macrophages. ( A ) Schematic representation of the extraction and identification of exosomes and the induction of macrophage polarization. Transmission electron microscopy ( B ), nanoparticle tracking analysis ( C ), and western blotting ( D ) were used to identify the morphology, particle size, and markers of exosomes. ( E ) Confocal laser scanning microscopy detected Dil-labeled exosomes (red) internalized by DAPI-labeled macrophages (blue). ( F – G ) Immunofluorescence analysis of the proportion of CD206 + cells in THP1 cells treated with exosomes. ( H – I ) Flow cytometry analysis of the proportion of CD68 + CD206 + cells in THP1 cells treated with exosomes. ( J – K ) qRT-PCR analysis of M1 markers (iNOS and TNF-α) and M2 markers (TGF-β, IL-10, and Arg-1) in THP1 cells treated with exosomes. ( L – N ) Transwell migration and invasion assays of GC cells (upper chamber) co-cultured with macrophages (lower chamber) ingesting exosomes

Article Snippet: THP-1 cells were differentiated into macrophages using 150 ng/mL phorbol 12-myristate 13-acetate (PMA, Sigma) for 24 h and subsequently co-cultured with cancer-derived exosomes or GC cells in 6-well plates with 0.4-μm membranes for 72 h. Harvested macrophages were converted into single-cell suspensions, stained with Elab Fluor 488 anti-human CD68 (Mouse, 1:20, ElabScience) and APC anti-human CD206 (Mouse, 1:20, ElabScience) antibodies, and analyzed for CD68 + CD206 + populations by flow cytometry (Accuri C6, BD).

Techniques: Derivative Assay, Extraction, Transmission Assay, Electron Microscopy, Western Blot, Confocal Laser Scanning Microscopy, Labeling, Immunofluorescence, Flow Cytometry, Quantitative RT-PCR, Migration, Cell Culture