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TAMs from HNSCC predominantly exhibit the M2 phenotype. A The morphological changes that occur when M0 macrophages were induced into TAMs by SCC25/CAL27-conditioned medium. The blue arrow indicated the emergence of pseudopodia in TAMs. B RT-qPCR was used to detect the mRNA levels of M2 phenotype markers ( FIZZ-1 , ARG-1 ) in TAMs. C Western blot was used to detect the protein levels of the M2 phenotype markers in TAMs (CD163, CD206). D Representative images of IF staining for CD163, CD206 and F-actin in macrophages induced by HNSCC conditioned medium, Scale bars = 50 μm. E Expression of classical M2 macrophage markers (CD206 and CD163) was evaluated by flow cytometry. F Western blot was performed to detect the expression of positive markers (TSG101, CD63, CD9) and negative marker (Calnexin) in the TAM-EVs. G Electron microscopy images of EVs were isolated from M0/TAM-CM. H <t>Nanoparticle</t> tracking analysis (NTA) of M0/TAM-sEVs. ns > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001
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high sensitivity flow cytometry for nanoparticle tracking analysis (nta)  (NanoFCM Inc)
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TAMs from HNSCC predominantly exhibit the M2 phenotype. A The morphological changes that occur when M0 macrophages were induced into TAMs by SCC25/CAL27-conditioned medium. The blue arrow indicated the emergence of pseudopodia in TAMs. B RT-qPCR was used to detect the mRNA levels of M2 phenotype markers ( FIZZ-1 , ARG-1 ) in TAMs. C Western blot was used to detect the protein levels of the M2 phenotype markers in TAMs (CD163, CD206). D Representative images of IF staining for CD163, CD206 and F-actin in macrophages induced by HNSCC conditioned medium, Scale bars = 50 μm. E Expression of classical M2 macrophage markers (CD206 and CD163) was evaluated by flow cytometry. F Western blot was performed to detect the expression of positive markers (TSG101, CD63, CD9) and negative marker (Calnexin) in the TAM-EVs. G Electron microscopy images of EVs were isolated from M0/TAM-CM. H <t>Nanoparticle</t> tracking analysis (NTA) of M0/TAM-sEVs. ns > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001
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TAMs from HNSCC predominantly exhibit the M2 phenotype. A The morphological changes that occur when M0 macrophages were induced into TAMs by SCC25/CAL27-conditioned medium. The blue arrow indicated the emergence of pseudopodia in TAMs. B RT-qPCR was used to detect the mRNA levels of M2 phenotype markers ( FIZZ-1 , ARG-1 ) in TAMs. C Western blot was used to detect the protein levels of the M2 phenotype markers in TAMs (CD163, CD206). D Representative images of IF staining for CD163, CD206 and F-actin in macrophages induced by HNSCC conditioned medium, Scale bars = 50 μm. E Expression of classical M2 macrophage markers (CD206 and CD163) was evaluated by flow cytometry. F Western blot was performed to detect the expression of positive markers (TSG101, CD63, CD9) and negative marker (Calnexin) in the TAM-EVs. G Electron microscopy images of EVs were isolated from M0/TAM-CM. H <t>Nanoparticle</t> tracking analysis (NTA) of M0/TAM-sEVs. ns > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001
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TAMs from HNSCC predominantly exhibit the M2 phenotype. A The morphological changes that occur when M0 macrophages were induced into TAMs by SCC25/CAL27-conditioned medium. The blue arrow indicated the emergence of pseudopodia in TAMs. B RT-qPCR was used to detect the mRNA levels of M2 phenotype markers ( FIZZ-1 , ARG-1 ) in TAMs. C Western blot was used to detect the protein levels of the M2 phenotype markers in TAMs (CD163, CD206). D Representative images of IF staining for CD163, CD206 and F-actin in macrophages induced by HNSCC conditioned medium, Scale bars = 50 μm. E Expression of classical M2 macrophage markers (CD206 and CD163) was evaluated by flow cytometry. F Western blot was performed to detect the expression of positive markers (TSG101, CD63, CD9) and negative marker (Calnexin) in the TAM-EVs. G Electron microscopy images of EVs were isolated from M0/TAM-CM. H <t>Nanoparticle</t> tracking analysis (NTA) of M0/TAM-sEVs. ns > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001
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TAMs from HNSCC predominantly exhibit the M2 phenotype. A The morphological changes that occur when M0 macrophages were induced into TAMs by SCC25/CAL27-conditioned medium. The blue arrow indicated the emergence of pseudopodia in TAMs. B RT-qPCR was used to detect the mRNA levels of M2 phenotype markers ( FIZZ-1 , ARG-1 ) in TAMs. C Western blot was used to detect the protein levels of the M2 phenotype markers in TAMs (CD163, CD206). D Representative images of IF staining for CD163, CD206 and F-actin in macrophages induced by HNSCC conditioned medium, Scale bars = 50 μm. E Expression of classical M2 macrophage markers (CD206 and CD163) was evaluated by flow cytometry. F Western blot was performed to detect the expression of positive markers (TSG101, CD63, CD9) and negative marker (Calnexin) in the TAM-EVs. G Electron microscopy images of EVs were isolated from M0/TAM-CM. H Nanoparticle tracking analysis (NTA) of M0/TAM-sEVs. ns > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001

Journal: Cellular and Molecular Life Sciences

Article Title: Tumor-associated macrophage-derived exosomal miR21-5p promotes tumor angiogenesis by regulating YAP1/HIF-1α axis in head and neck squamous cell carcinoma

doi: 10.1007/s00018-024-05210-6

Figure Lengend Snippet: TAMs from HNSCC predominantly exhibit the M2 phenotype. A The morphological changes that occur when M0 macrophages were induced into TAMs by SCC25/CAL27-conditioned medium. The blue arrow indicated the emergence of pseudopodia in TAMs. B RT-qPCR was used to detect the mRNA levels of M2 phenotype markers ( FIZZ-1 , ARG-1 ) in TAMs. C Western blot was used to detect the protein levels of the M2 phenotype markers in TAMs (CD163, CD206). D Representative images of IF staining for CD163, CD206 and F-actin in macrophages induced by HNSCC conditioned medium, Scale bars = 50 μm. E Expression of classical M2 macrophage markers (CD206 and CD163) was evaluated by flow cytometry. F Western blot was performed to detect the expression of positive markers (TSG101, CD63, CD9) and negative marker (Calnexin) in the TAM-EVs. G Electron microscopy images of EVs were isolated from M0/TAM-CM. H Nanoparticle tracking analysis (NTA) of M0/TAM-sEVs. ns > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: The diameter of the sEV was measured using high-sensitivity flow cytometry for nanoparticle tracking analysis (NTA; Nanofcm, Fujian, China).

Techniques: Quantitative RT-PCR, Western Blot, Staining, Expressing, Flow Cytometry, Marker, Electron Microscopy, Isolation