S2745

Geckos with well-regenerated blastema at day 12 post autotomy were prepared. The anesthetized ones were sterilized with 70% EtOH and the blastema tissues were dissected out and digested in 0.25% trypsin-EDTA buffer at 30 °C for 10 min. Then cells were harvested and resuspended in 3 mL DMEM/F12 (10-092-CV, Thermo Fisher Scientific) containing 10% FBS, 2 mM glutamine (C0212, Beyotime, Nantong, China) and Penicillin-Streptomycin-Amphotericin B solution (100X, C0224, Beyotime, Nantong, China). Cells then were plated in 8-cm² cell-culture dish under 30 °C and 5% CO2. The cells of passage 2 were gathered by conventional trypsin dislocation and allocated into a 6-well plate with 3 × 10⁵ cells per well for 12 h. The Wnt/β-catenin signaling agonist SKL2001 (40 μM, S8320, Selleck, Houston, TX) or CHIR98014 (8 μM, S2745, Selleck, Houston, TX) were applied into culture medium for additional 24 h before assays. The Akt antagonist MK-2206 (8 μM, S1078, Selleck, Houston, TX) were administrated as above.

The tails were amputated at the 6th caudal vertebrae to set up the regeneration models. Akt inhibitor MK-2206 (5 mg/kg body weight (BW) S1078, Selleck, Houston, TX) was administered after autotomy by i.p. injection every other day. BrdU solvent (20 μL of 20 mM solution, B5002, Sigma, Darmstadt, Germany) was administered on the last 2 days before sample collection. The samples were collected on the 7th day or other time point indicated. For the treatment of mTOR inhibitor (RAD001, Torin1) and AMPK activator (A769662, Selleck, Houston, TX), the gecko models were administered RAD001 (10 mg/kg BW, S1120, Selleck, Houston, TX), Torin1 (20 mg/kg BW, S2827, Selleck, Houston, TX) and A769662 (30 mg/kg BW, S2697, Selleck, Houston, TX) individually or together by i.p. injection post autotomy every other day. For the treatment of Wnt agonist, CHIR98014 was administered (5 mg/kg BW, S2745, Selleck, Houston, TX) by i.p. injection post autotomy every other day. The BrdU was administered as mentioned above. For treatment with the Wnt inhibitor, IWP-4 (2 mg/kg BW, SML1114, Sigma, Darmstadt, Germany) or XAV939 solution (40 mg/kg BW, S1180, Selleck, Houston, TX) was administered after amputation by i.p. injection every other day.