Fig. 5

miR-99b-5p delivered by MSC-EVs inhibited HG-induced proliferation and ECM accumulation by promoting mTOR-mediated autophagy in mMCs. (A) Cell viability of mMCs was measured by a CCK-8 assay under conditions of NG, MA, HG, and HG with EVs-NC or HG with EVs-KD for 24, 48 and 72 h. (B, C) Representative Western blot images and quantitative analysis of PCNA expression in mMCs subjected to different treatments. β-actin was used as a loading control. The full-length blots are presented in Supplementary Fig. 9. (D, E) EdU incorporation assay in mMCs subjected to different treatments and quantification of the number of EdU-positive cell nuclei (green)/total number of cell nuclei (blue). Nuclei were counterstained with Hoechst 33,342. Scale bar, 100 μm. (F, G) Immunofluorescence staining and quantification of α-SMA (green), fibronectin (red) and Col IV (red) expression in mMCs subjected to different treatments. Nuclei (blue) were counterstained with DAPI. Scale bar, 100 μm. (H, I) Representative western blot images and quantitative analysis of mTOR, p-mTOR, LC3II/LC3I and p62 expression in mMCs subjected to different treatments. β-actin was used as a loading control. The full-length blots are presented in Supplementary Fig. 9; n = 3. The data are expressed as the means ± SEMs. Statistical analysis was performed via one-way ANOVA with Tukey’s multiple comparison tests. ^*P < 0.05 versus NG; ^#p < 0.05 versus HG. ^&p < 0.05 versus HG + EV-NC. Abbreviations: NG, normal glucose. HG, high glucose