Heparan sulfate co-immobilized with cRGD ligands and BMP2 on biomimetic platforms promotes BMP2 mediated osteogenic differentiation. Figure 1 c and g
BMP2-SMAD signaling is enhanced by increasing the surface density of cRGD peptides likely through increased engagement of integrins at the cell membrane. The phosphorylation of SMAD 1/5/9 is not enhanced if C2C12 cells are plated on increasing cRAD peptide concentration. Figure 1c: Increasing surface concentration of cRGD, and therefore the integrins engagement at the membrane, enhances cell response to sBMP2. Western blot analysis of p-SMAD 1/5/9 levels in C2C12 cells plated on surfaces presenting three different cRGD surface concentrations and stimulated with sBMP2 (6 nM). As control, C2C12 cells were plated on platforms presenting only SAv and on a plastic petri-dish (TCPS). One hour after plating cells were lysed and the level of p-SMAD 1/5/9 plotted and normalized to the housekeeping gene GAPDH. Two experiments realized and used for quantification, as well as the experiment shown in Figure 1c of the paper Sefkow-Werner et al., 2020, (submitted), are shown here. The quantification of the three independent experiments shows the average ± SEM in Figure 1d of the cited paper. Figure 1g: Western blot of p-SMAD 1/5/9 expression of C2C12 cells plated for one hour on cRAD platforms and exposed to the same concentration of sBMP2 (6 nM). As control, C2C12 were plated on a plastic petri-dish (TCPS) with or without sBMP2. One hour after plating cells were lysed and the level of p-SMAD 1/5/9 plotted and normalized to the housekeeping gene GAPDH. The two experiments realized and used, together with the blot shown in Figure 1g of the paper Sefkow-Werner et al., 2020 (submitted), are shown here. The quantification of the average ± SEM of the three independent experiments is shown in Figure 1h.