(A, B) PG (green) and nuclei (magenta: DAPI) show that the morphology of the gland is intact in late L3 larvae with reduced expression of vvl, kni, or mld in the PG. PG cell and nucleus size of L3 larvae (measured as the diameter) are reduced when vvl and kni are knocked down (n = 11). RNAi was induced using the Gal80ts;phm>96 hours AEL corresponding to the late L2 stage at 18°C. The PG was analyzed after two days at 29°C when control larvae are in the wandering stage. (C) Knock down of vvl and kni results in decreased expression of torso (tor), 4EBP and S6K in dissected ring glands containing the PG. Likewise, 4EBP and S6K are down regulated in ring glands of mld-RNAi animals that also exhibit reduced expression of the InR. Contrary, akt is increased in vvl-RNAi animals (n = 4). (D) Loss of vvl, kni or mld results in a strong increase of npc1a expression in the ring gland. In C and D, expression was measured in L3 larvae raised as described in A and B (n = 4). Error bars indicate s.e.m. *P<0.05, **P<0.01, ***P<0.001, versus the Gal80ts;phm>+ control.