Figure 6. Effects of 14-3-3 RNAi on Tor targets and the suppression by CycE.

(a) Effects of 14-3-3 RNAi on S6k and Thor. Either one of 14-3-3 isoforms or both 14-3-3s were knocked down in S2 cells. Single or double knockdown of 14-3-3s reduced the level of pS6k (Thr 398) but does not affect the level of S6k protein. Knockdown of both 14-3-3s, but not one isoform, causes slight reduction of Thor. In contrast, pThor (Thr 37/46) is strongly reduced by single or double knockdown of 14-3-3s. (b–d) Loss of 14-3-3s results in reduction of CycE level. (b) 14-3-3ɛ mutant clones induced by ey-flp are marked by the absence of GFP. Mutant clones show strong reduction of CycE (c). (e–g) 14-3-3ζ mutant clones induced by hs-flp are marked by the absence of Arm-lacZ. CycE is reduced in mutant clones (f). (h–m) Effects of CycE in eye. Control with one copy of ey-Gal4 (h) or overexpression of CycE (i) shows normal eye. (j) Double knockdown of Tctp and 14-3-3 results in small and rough eye phenotype (<40% of wild-type eye size). (k–m) Overexpression of CycE rescues the small-eye phenotype resulting from double knockdown of Tctp and 14-3-3. The rescue is varied, ranging from strong (S, 81–100% of wild-type size) (k), intermediate (I, 41–80%) (l) to no rescue (N, 40% or lower) (m). (n) Quantification of rescue shown in k–m. More than 60 flies with the genotype of ey>Tctp RNAi/CycE GFP; 14-3-3 RNAi/+ were scored. (o–r) CycE suppresses the wing phenotypes of double knockdown of Tctp and 14-3-3. Control with one copy of MS1096-Gal4 (o) or CycE overexpression (p) does not affect wing development. (q) Knockdown of both Tctp and 14-3-3 results in small and wrinkled wing. (r) Overexpression of CycE partially rescues the double-knockdown phenotype in 100% of tested flies. Scale bar, 100 μm (b–g); 200 μm (h–m); 400 μm (o–r).