S1120

Cells were plated at a density of 2000 cells/well in 96-well plates, and cell viability and cytotoxicity were measured according to the protocol (Yeasen Biotechnology, Shanghai, China). When performing the drug sensitivity analysis, the cells were incubated with 100 μL of fresh medium containing vehicle (DMSO) or drugs for 48 h and were assayed for cell viability measurement. The following drugs were used at the indicated concentrations: axitinib (1 μM, Selleck, S1005), sunitinib (5 μM, Selleck, S7781), pazopanib (5 μM, Selleck, S3012) and everolimus (25 nM, Selleck, S1120). For the EdU assays, post transfection for 48 h, cells were seeded in 12-well plates at a density of 8 × 10⁴ cells per well. The cells were then incubated with a medium containing 50 μM EdU for a period of 2 h. Subsequently, the cells were fixed with 4 % paraformaldehyde at room temperature for a duration of 20 min. This was followed by staining the cells with Hoechst reagent and Apollo staining solution. The percentage of EdU-positive cells was calculated under a microscope in five randomly selected fields.

Everolimus, temsirolimus and rapamycin were purchased from Selleck Chemicals (S1120, S1044 and S1039) (Houston, TX). Growth inhibition was assessed by the MTS assay to examine the effect of everolimus, temsirolimus and rapamycin on SCLC cell lines as previously described [16 (link)]. Cell suspensions (5,000 cells/well) were seeded into 96-well plates and increasing concentrations of everolimus, temsirolimus and rapamycin (0, 0.001, 0.01. 0.1, 1.0, 10 and 100 μM) were added. After incubation at 37 °C for 72 h, MTS was added to each well and incubated at 37 °C for 2 h, after which absorbance was measured at a test wavelength of 490 nm using a microplate reader (Dynatech MR7000, Dynatech, Billinghurst, UK). The IC50 value was defined as the concentration of everolimus, temsirolimus or rapamycin needed for 50% reduction of growth and was calculated by SigmaPlot12 (HULINKS, Inc, Tokyo, Japan). Each experiment was performed independently three times. The corrected absorbance of each sample was calculated and compared with that of the untreated control.

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.