Fig. 4: 5TGM1 sEVs induce apoptosis and inhibit differentiation of MC3T3-E1 cells.

a–d. MC3T3-E1 cells were cultured with 5TGM1/5T33vt concentrated conditioned medium (CCM) or 5TGM1/5T33vt sEVs (100 µg/ml) in serum-free medium. Control = serum-free medium. a. Relative viability after 48 h was determined by a Cell Titer Glo® luminescence assay. b. Relative proliferation was determined after 24 h with a BrdU incorporation assay. c. Relative caspase activity determined by CaspaseGlo after 24 h. d. Living MC3T3-E1 cells after culture for 24 h determined by Flow Cytometry analysis (AnnV/7AAD negative cells). e Relative mRNA expression was determined by qRT-PCR for RUNX2 in undifferentiated MC3T3-E1 cells. Osterix (Osx), Collagen 1A1 (Col1A1) and Alkaline phosphatase (ALP) expression were compared in differentiated MC3T3-E1 cells after 24 h of culture with the three previously described conditions. f Relative ALP activity was measured after 24 h by an ALP yellow liquid substrate assay. ALP activity was normalized to total amount of protein. g Western blot analysis from MC3T3-E1 cell lysates after 24 h of culture with either serum-free medium, 5TGM1 CCM or 5TGM1 sEVs, representing downregulation of Wnt signaling pathway, Runx2 expression, and loss of phospho-Akt. h Western blot analysis shows the presence of DKK1 on 5TGM1 sEVs and in the 5TGM1 cell lysate (5TGM1 cells). i qRT-PCR shows upregulation of DKK1 in differentiated osteoblasts cultured with 5TGM1 CCM or sEVs for 24 h. Control = serum-free medium. j Visualization of uptake of 5TGM1 sEVs by MC3T3-E1 cells after 24 h by confocal microscopy. Nuclei and membranes of MC3T3-E1 cells were stained by Hoechst and WGA respectively. 5TGM1 sEVs were labeled with DiO-membrane staining. Bars represent mean ± SD. Experiments were repeated at least three times independently. * = p < 0.05