Tgfβ receptor 1 inhibitor

Once VEH- or EDC-MMSCs reached 80% confluence, they were treated with human recombinant TGFβ1 (10 ng/ml, 7754-BH, R&D Systems,) for 48 h or with TGFβ Receptor I inhibitor (2 µM, LY-364947, L6293, Sigma, St. Louis, MO, USA) for 24 h, respectively. The vehicle used to dissolve TGFβ1 was 4 mM HCl (SA49, Thermo Fisher Scientific, Waltham, MA, USA) containing 0.1% bovine serum albumin (BSA, A3294, Sigma, St. Louis, MO, USA). Mature human TGFβ1 shares 99% amino acid identity with rat TGFβ1, and it demonstrated cross-species activity [36 ]. Dimethyl sulfoxide (DMSO, 472301, Sigma, St. Louis, MO, USA) was used as a VEH to dissolve the TGFβ Receptor I inhibitor (final concentration < 0.1%). VEH- and EDC-treated MMSCs were washed with PBS, trypsinized, and centrifuged at 500×g for 5 min. The supernatant was discarded, and the pellets were snap-frozen and stored at − 80 °C for RNA isolation.

Once VEH- or EDC-MMSCs reached 80% confluence, they were treated with human recombinant TGFβ1 (10 ng/ml, 7754-BH, R&D Systems,) for 48 h or with TGFβ Receptor I inhibitor (2 µM, LY-364947, L6293, Sigma, St. Louis, MO, USA) for 24 h, respectively. The vehicle used to dissolve TGFβ1 was 4 mM HCl (SA49, Thermo Fisher Scientific, Waltham, MA, USA) containing 0.1% bovine serum albumin (BSA, A3294, Sigma, St. Louis, MO, USA). Mature human TGFβ1 shares 99% amino acid identity with rat TGFβ1, and it demonstrated cross-species activity [36 ]. Dimethyl sulfoxide (DMSO, 472301, Sigma, St. Louis, MO, USA) was used as a VEH to dissolve the TGFβ Receptor I inhibitor (final concentration < 0.1%). VEH- and EDC-treated MMSCs were washed with PBS, trypsinized, and centrifuged at 500×g for 5 min. The supernatant was discarded, and the pellets were snap-frozen and stored at − 80 °C for RNA isolation.