Pd0325901 s1036

Cell lines, established from tumors arising in irradiated Nf1^+/− F1 mice (17 (link)). Early passage stocks were established for all cell lines and experimental cells were drawn from these stocks approximately every twenty to thirty passages. Mycoplasma testing was done approximately yearly. Inhibitors were purchased from Selleck Chemicals (PD0325901 S1036, Everolimus (RAD001) S1120, Dactolisib (BEZ235) S1009, PD184352 (CI-1040) S1020, Torkinib (PP242) S2218, PIK-75 HCl S1205, Tozasertib (VX680) S1048, Barasertil (AZD1152) S1147, Alisertib (MLN8237) S1133, Binimetinib (MEK162) S7007, Trametinib (GSK1120212) S2673, Rapamycin (Sirolimus) S1039). Drug-resistant cell lines 989 PD_R, 989 RAD_R, 881 PD_R and 881 RAD_R were derived from each original parental cell line by continuous exposure to PD0325901 or RAD001. Cell lines 989 and 881 were exposed to increasing concentrations (from 25 nM to 500 nM) of PD0325901 or RAD001 for six months. Resistant cell lines were continuously maintained in medium supplemented with inhibitor. For drug washout experiments, cells were washed twice with PBS, inhibitors were withdrawn for 12 days and further analyses performed.

Crosses were set up with esg-GAL4, tub-GAL80^TS, UAS-GFP and UAS-yki^3SA or UAS-dERK^SEM; UAS-yki^3SA at 18°C to inactivate GAL4. 4-day-old adult progenies were placed at 29°C to induce the transge nes. For the in vivo RNAi screening of ligands in yki^3SA-tumor guts, different RNAi lines were crossed to esg-GAL4, tub-GAL80^TS, UAS-GFP/CyO; UAS-yki^3SA/TM6B at 18 °C . Virgin progenies were maintained at 18 °C for at least 8 d ays and then switched to 29 °C to induce transgenes. Lipid and sugar levels, cl imbing speed, and muscle morphology, gut morphology, and bloating phenotypes were all measured after switching to 29 °C for 8 days. For drug treatment, flies were transferred onto food containing inhibitors at day 0 (simultaneously with tumor induction) or day 4 (after tumor formation). MEK/ERK inhibitors PD0325901 (S1036) and Trametinib (S2673) were purchased from Selleckchem.