Dorsomorphin compound c

Following steady state, cells were rinsed and incubated in Ca²⁺-free S-MEM supplemented with 5% dialyzed FBS ([Ca²⁺], 5 μM) for 16 h before being switched back to normal medium (α-MEM; [Ca²⁺], 1.8 mM) for the indicated times. Compounds STO-609 (50 μM; Sigma) and dorsomorphin/compound C (50 μM; Sigma) were used as CaMKK and AMPK inhibitors, respectively. Increasing concentrations of dorsomorphin/compound C were tested in order to fully block AMPK activation (Supplementary Figure, panel A, available online at https://doi.org/10.1155/2017/9717353). Alternatively, MDCK cells were exposed to MSC-conditioned medium for 24 h, before exposure to S-MEM for 16 h. The Ca²⁺ switch was then realized using MSC-conditioned α-MEM for the indicated times.

Neferine (C38H44N2O6) was purchased from Sigma (St. Louis, MO, USA) and solvated in dimethyl sulfoxide. 3T3-L1 preadipocytes were acquired from the ATCC (Manassas, VA, USA). Cell growth medium (DMEM), bovine calf serum (BCS), trypsin, fetal bovine serum (FBS), and insulin were supplied by Thermo Fisher (San Jose, CA, USA), while antibiotic-antimycotic solution, 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), dorsomorphin (Compound C), 3-isobutyl-1-methylxanthine (IBMX), and dexamethasone (DEX) were procured from Sigma (St. Louis, MO, USA).

The autophagy activator (AA) Rapamycin (V900930, Sigma Aldrich) and the autophagy inhibitor (AI) dorsomorphin/Compound C (P5499, Sigma Aldrich) were dissolved and stored at 4 C. Group designs and schematic protocol of in vitro and in vivo experiments are shown as follows (Figure 1).
HCECs in the control group were treated with PBS; HCECs in the Exo group were treated with 1 × 10⁶/μl hucMSC-Exos; HCECs in the AA group were treated with 50 nM Rapamycin; HCECs in the AI group were treated with 5 μM Compound C; HCECs in the Exo + AA group were treated with 1 × 10⁶/μl hucMSC-Exos and 50 nM Rapamycin; and HCECs in the Exo + AI group were treated with 1 × 10⁶/μl hucMSC-Exos and 5 μM Compound C.
Normal corneas in the control group were treated with PBS; injured corneas in the CI + PBS group were treated with PBS; injured corneas in the CI + L-Exo group were treated with 1 × 10⁵/μl hucMSC-Exos; injured corneas in the CI + M-Exo group were treated with 1 × 10⁶/μl hucMSC-Exos; injured corneas in the CI + H-Exo group were treated with 1 × 10⁷/μl hucMSC-Exos; injured corneas in the CI + Exo + AA group were treated with 1 × 10⁶/μl hucMSC-Exos and 10 μM Rapamycin; and injured corneas in the CI + Exo + AI group were treated with 1 × 10⁶/μl hucMSC-Exos and 50 μM Compound C.