Figure 5. 14-3-3s are required for the interaction between Tctp and Rheb and organ development.

(a) Knockdown of 14-3-3s in S2 cells shows depletion of 14-3-3s levels after 6 days of RNAi treatment. ɛi, ζi and ɛi+ζi indicate RNAi for 14-3-3ɛ, 14-3-3ζ and both isoforms, respectively. Levels of Tctp and Rheb were not noticeably affected by 14-3-3 depletion. The treatment performed to detect Tctp, Rheb, and actin level was double knockdown of both isoforms of 14-3-3. (b) 14-3-3 depletion affects the interaction between V5-Tctp and V5myc-Rheb after 6 days of RNAi treatment. Anti-Myc antibody was used for IP. Tctp was detected by anti-Tctp antibody. Depletion of both isoforms abolished the Tctp–Rheb interaction. (c–j) Double knockdown of 14-3-3s causes loss of targeted tissues. ey-Gal4 control (c) 14-3-3ɛ RNAi (d) and 14-3-3ζ RNAi (e) show normal pattern of eye-head in pupae. Knockdown of both 14-3-3 isoforms causes pupal lethality with loss of eye and head (f). (g–j) Pattern of Elav staining in larval eye discs from indicated genotypes. Knockdown of 14-3-3ɛ or 14-3-3ζ has no obvious effect on eye disc development (h,i). Knockdown of both isoforms results in small disc (the area marked with dotted line) with no retinal differentiation (j). (k–n) Double knockdown of 14-3-3s results in reduced proliferation. MS1096-Gal4 control (k) 14-3-3ɛ RNAi (l) and 14-3-3ζ RNAi (m) show normal level of PH3 staining in wing disc. Knockdown of both 14-3-3 isoforms causes strong reduction of PH3 in the wing pouch (circled area) (n). (o–r) Caspase activation marked by anti-Caspase 3 (Cas-3) staining is not detected in MS1096-Gal4 control (o) 14-3-3ɛ RNAi (p) and 14-3-3ζ RNAi (q). Double knockdown of both 14-3-3 isoforms results in strong activation of Cas-3 (r). Scale bar, 300 μm (c–f); 100 μm (g–j); 100 μm (k–r).