Norleual

L6 rat myoblasts and C2C12 mouse myoblasts were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Thermo Fisher Scientific; #31966021) containing l-alanyl-l-glutamine (GlutaMAX), 10% FBS, and 1% penicillin-streptomycin. Myoblasts were kept at a subconfluent level (< 80%) to prevent the loss of myoblastic component as the cells were passaged. In co-culture experiments where proliferation was studied, myoblasts were seeded in normal growth medium and then switched to low-serum medium (DMEM supplemented with 1% FBS) 24 h before exposing them to SVF cells. SVF cells were added at a 1:1, 1:2, or 1:5 ratio to myoblasts in 0.1 μm PET transwell membrane inserts (Corning; #353104) and co-cultured for up to 5 days. In pharmacological inhibitor experiments, MAP kinase kinase (MEK) inhibitor (25 μM; Calbiochem; #513001), Atropine (10⁻⁵ M, Sigma-Aldrich; #A0132), or Norleual (100 pM, Tocris; #5369) were added to cell cultures 2 h prior to adding the SVF cells. In cases where HGF (PeproTech; #100-39) was used the concentration varied from 5 to 30 ng/ml.

All animal procedures were approved by Duke University DLAR following the protocol A286‐15‐10. NOD.Cg‐Prkdc^scidil2rg^tm1Wjl/SzJ (NSG) mice and BALB/c mice used in this study were 8 weeks old. To test DPP4 inhibition in vivo, three days after spleen injection, Sitagliptin (Millipore Sigma, Cat#Y0001812) was administered to mice twice a day via oral gavage at 250 mg kg⁻¹.^[64] To test HGF inhibition in vivo, three days post spleen injection, Norleual (Tocris, Cat#5369) was administered to mice i.p. twice a day at 1 mg kg⁻¹.^[65]