Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Involvement of miRNA-204 carried by the exosomes of macrophages in the AT2 receptor-mediated improvement of vascular calcification

doi: 10.1007/s00018-025-05703-y

Figure Lengend Snippet: Effects of macrophages on the AT2R-mediated improvement of vascular calcification. A Effects of macrophages on RASMC calcification after β-glycerophosphate (10 mmol/L) and/or C21 (10 µmol/L) treatment for 14 days. Representative images of calcified nodules evaluated by Alizarin red staining (original magnification ×100). The results of the quantitative analysis of Alizarin red staining are shown as the OD value. B, C The expression of calcification-related proteins associated with the calcification of RASMCs cocultured with macrophages after C21 treatment for 14 days. Photos of BMP-2 and OCN expression determined by immunofluorescence staining (original magnification ×100). The results of the quantitative analysis of the immunofluorescence staining are shown as the relative fluorescence intensity. D The expression of calcification-related signalling pathway proteins in RASMCs cocultured with macrophages after C21 treatment for 14 days. The expression of Wnt3a and β-catenin was determined by Western blotting. The values are the means ± S.E.M.s n = 3 for each group. *p < 0.05, **p < 0.01 vs. CON. ##p < 0.01 vs. Pi. &&p < 0.01 vs. CON (coculture). ++p < 0.01 vs. Pi (coculture). Co-culture RASMCs cocultured with macrophages

Article Snippet: Some cells were cultured with or without 10 mmol/L β-glycerophosphate (Sigma, G9422), 0.25 mmol/L ascorbic acid (Sigma, A7506) and/or 10 µmol/L C21 (MedChemExpress, HY-100113) treatment.

Techniques: Staining, Expressing, Immunofluorescence, Fluorescence, Western Blot, Co-Culture Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Involvement of miRNA-204 carried by the exosomes of macrophages in the AT2 receptor-mediated improvement of vascular calcification

doi: 10.1007/s00018-025-05703-y

Figure Lengend Snippet: Polarization and inflammation of macrophages in AT2R-mediated improvement of vascular calcification. A–C Polarization of macrophages after β-glycerophosphate and/or C21 treatment for 14 days. Representative images of CD68, CD11c and CD206 expression determined by immunofluorescence staining (original magnification ×100). D Results from the quantitative analysis of immunofluorescence staining are shown as the relative fluorescence intensity. The polarization of macrophages was evaluated by the ratio of the relative fluorescence intensity of M1 to that of M2. E–H The expression of proinflammatory and anti-inflammatory markers released by macrophages after β-glycerophosphate and/or C21 treatment for 14 days. Representative images of IL-1β, TNF-α, IL-10 and TGF-β expression, as determined by immunofluorescence staining (original magnification × 100). The results of the quantitative analysis of the immunofluorescence staining are shown as the relative fluorescence intensity. The values are the means ± S.E.M.s n = 3 for each group. *p < 0.05, **p < 0.01 vs. CON. #p < 0.05, ##p < 0.01 vs. Pi

Article Snippet: Some cells were cultured with or without 10 mmol/L β-glycerophosphate (Sigma, G9422), 0.25 mmol/L ascorbic acid (Sigma, A7506) and/or 10 µmol/L C21 (MedChemExpress, HY-100113) treatment.

Techniques: Expressing, Immunofluorescence, Staining, Fluorescence

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Involvement of miRNA-204 carried by the exosomes of macrophages in the AT2 receptor-mediated improvement of vascular calcification

doi: 10.1007/s00018-025-05703-y

Figure Lengend Snippet: Exosomes secreted by macrophages in AT2R-mediated improvement of vascular calcification. A–C Identification of macrophage-secreted exosomes after β-glycerophosphate and/or C21 treatment for 72 hours. Size‒concentration curve of macrophage-secreted exosomes drawn after nanoparticle tracking analysis (NTA). The expression of surface markers of macrophage-secreted exosomes (ALIX, TSG101 and CD9) was determined by Western blotting. Representative images of macrophage-secreted exosomes taken via electron microscopy (original magnification × 30000, × 49000). D, E The relative expression of macrophage-secreted exosomal miRNA-204-5p determined by qPCR after β-glycerophosphate and/or C21 treatment with or without miRNA-204-5p inhibitor stimulation for 72 hours. The values are the means ± S.E.M.s n = 3 for each group. *p < 0.05, **p < 0.01 vs. CON. ##p < 0.01 vs. Pi. &&p < 0.01 vs. C21. Inhibitor miRNA-204-5p inhibitor

Article Snippet: Some cells were cultured with or without 10 mmol/L β-glycerophosphate (Sigma, G9422), 0.25 mmol/L ascorbic acid (Sigma, A7506) and/or 10 µmol/L C21 (MedChemExpress, HY-100113) treatment.

Techniques: Expressing, Western Blot, Electron Microscopy

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Involvement of miRNA-204 carried by the exosomes of macrophages in the AT2 receptor-mediated improvement of vascular calcification

doi: 10.1007/s00018-025-05703-y

Figure Lengend Snippet: Effects of exosomal miRNA-204-5p on AT2R-mediated improvements in RASMC calcification. A Effects of exosomal miRNA-204-5p on RASMC calcification after β-glycerophosphate and/or C21 treatment for 14 days. Representative images of calcified nodules evaluated by Alizarin red staining (original magnification ×100). The results of the quantitative analysis of Alizarin Red S staining are shown as the OD value. B Effects of exosomal miRNA-204-5p on the expression of calcification-related signalling pathway proteins during RASMC calcification after β-glycerophosphate and/or C21 treatment for 14 days. The expression of Wnt3a and β-catenin was determined by Western blotting. C, D Effects of exosomal miRNA-204-5p on the expression of calcification-related proteins involved in RASMC calcification after β-glycerophosphate and/or C21 treatment for 14 days. Photos of BMP-2 and OCN expression determined by immunofluorescence staining (original magnification ×100). The results of the quantitative analysis of the immunofluorescence staining are shown as the relative fluorescence intensity. E Effects of exosomal miRNA-204-5p on the expression of RUNX2 during RASMC calcification after β-glycerophosphate and/or C21 treatment for 14 days. The expression of RUNX2 was determined by Western blotting. F The interaction between miRNA-204-5p and RUNX-2 mRNA was determined via a luciferase activity assay. The results of the quantitative analysis of the luciferase activity assay are shown as the relative luciferase activity. The values are the means ± S.E.M.s n = 3 for each group. *p < 0.05, **p < 0.01 vs. CON. ## p < 0.01 vs. Pi. &p < 0.05, &&p < 0.01 vs. CON (Inhibitor). ++p < 0.01 vs. Pi (Inhibitor). Inhibitor miRNA-204-5p inhibitor, NC negative control of miRNA-204-5p, WT the wild-type of RUNX2 mRNA, MUT the mutant versions of RUNX2 mRNA

Article Snippet: Some cells were cultured with or without 10 mmol/L β-glycerophosphate (Sigma, G9422), 0.25 mmol/L ascorbic acid (Sigma, A7506) and/or 10 µmol/L C21 (MedChemExpress, HY-100113) treatment.

Techniques: Staining, Expressing, Western Blot, Immunofluorescence, Fluorescence, Luciferase, Activity Assay, Negative Control, Mutagenesis