I0516

Glucose concentration in Drosophila hemolymph and brains was measured using the Infinity Glucose Hexokinase reagent (Thermo scientific). To measure glucose in the hemolymph, samples of 10 larvae were washed in NaCl 0.7%, 0.1% Triton X-100 and then in d-H₂O. The hemolymph of these larvae was collected and its glucose content measured following the protocol of Rulifson et al. [38] (link). The values reported in Figure 2A are the means ±SE of 8 samples of 10 larvae. To measure glucose in brains, samples of 20 brains were placed in 40 µl of 10⁻³ M EDTA, 10⁻²M KH₂PO₄, and the complete protease inhibitor cocktail (Roche), mechanically homogenized and then centrifuged at 14,000 rpm for 10 min. The supernatant was collected with a micropipette and used for glucose measurement according to Rulifson et al. [38] (link). The measures reported in Figure 2B are the means ±SE of 4 samples of 20 brains.
Insulin stimulation of Akt phosphorylation was performed using recombinant human insulin (Sigma, I0516). Before stimulation, larvae were starved for 5 hours in an empty vial humidified with a drop of saline devoid of FBS. We then followed the protocol described by Musselman et al. [41] (link). However, at the end of the insulin stimulation procedure, instead of larvae, we homogenized samples of 20 isolated brains, which were then used for Western blotting analysis.

HeLa cells were transduced with three lentiviral shRNA pools containing 2,833 shRNAs against 616 E3 ligase genes or NS shRNA in triplicate at 0.2 multiplicity of infection (MOI) to prevent superinfection and to ensure that each cell received no more than one shRNA. After infection, HeLa cells were selected with puromycin (0.2 μg ml⁻¹) for 7 days to enrich for HeLa cells expressing shRNA. After puromycin selection, HeLa cells were grown in serum-free DMEM containing trace elements (D0547, Sigma) supplemented with 50 ng ml⁻¹ EGF (E9644, Sigma), 20 ng ml⁻¹ FGF (SRP3043, Sigma), 100 nM hydrocortisone (H0888, Sigma), 0.5 μg ml⁻¹ transferrin (T1147, Sigma), 5 ng ml⁻¹ selenium (S5261, Sigma), 0.5 μg ml⁻¹ fibronectin (F1141, Sigma) and 0.05 μg ml⁻¹ insulin (I0516, Sigma). Media was changed every 3 days and cells were split at a 1:4 ratio every 7 days. After four passages all cells carrying NS and shRNAs from pool 2 were dead. Surviving cells from pools 1 and 3 were collected and genomic DNA was isolated. The integrated shRNAs were PCR-amplified using primers specific to the shRNA vector (pLKO.1) and listed in Supplementary Table 3. Samples were sequenced using primer SP6 (Supplementary Table 3) to identify candidate shRNAs.