Fig 2: Knockdown NBR2 in HCT116 and SW480 cells and the detected expression of glucose starvation-induced changes in protein phosphorylation in AMPK/mTOR signaling. A and B, Reverse transcription-PCR showed the level of NBR2 in both HCT116 and SW480 cells after transfection with shNBR2. C-F, Western blotting protein bands and quantitative analysis showed changes in the phosphorylation levels of AMPK, ACC, S6K, and S6 proteins in both HCT116 and SW480 cells treated with 0 or 25 mM glucose for 24 hours. Three independent experiments were performed, and the results are presented as the mean ± standard deviation. *P < .05. AMPK indicates adenosine monophosphate-activated protein kinase; RT-PCR, reverse transcription-polymerase chain reaction.

Fig 3: 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) 14-3-3? mutant clones induced by ey-flp are marked by the absence of GFP. Mutant clones show strong reduction of CycE (c). (e–g) 14-3-3? 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).

Fig 4: RpL10Ab suppresses TOR pathway activity.(a) Diagram showing the components of the TOR pathway assayed in the experiments. Orange and blue represent positive and negative regulators of TOR pathway respectively. (b) Western blots showing the amounts of pS6K, pAKT, S6K, AKT and Actin proteins in ovaries from fed Oregon R and RpL10Ab[CB02653]/+ F0 flies. (c) Quantification of the western blots shown in (b). n = 3 biological replicates. *p<0.05. ns: p>0.05. (d) % of starvation-resistant stage 14 oocytes in RpL10Ab[C03865]/+ and RpL10Ab[CB02653]/+ F0 mothers compared to F0 control. *: p<0.05, **: p<0.01. (e) Western blots showing the amounts of pS6K, pAKT, S6K, AKT and Actin proteins in ovaries from fed Oregon R and MTD-Gal4>FLAG-RpL10Ab F0 flies. (f) Quantification of the western blots shown in (e). n = 3 biological replicates. *p<0.05. **p<0.01. ns: p>0.05. (g) Western blots showing the co-immunoprecipitation between TSC2 and RpL10Ab in S2 cells. (h) Quantification of the western blots shown in (g). n = 3 biological replicates. *p<0.05. **p<0.01. (I) Western blots showing the levels of TSC2 protein in control versus S2 cells overexpressing FLAG-RpL10Ab. (j) Quantification of the western blots shown in (I). n = 3 biological replicates. **p<0.01. (k, l) % of starvation-resistant stage 14 oocytes in the indicated genotypes, indicating a genetic interaction between RpL10Ab and gig and rheb. *: p<0.05, **: p<0.01. (m) A genetic pathway showing the involvement of RpL10Ab in the TOR pathway in the regulation of starvation-induced stage 14 oocyte loss.

Fig 5: CCT complex physically interacts with TOR components. a Co-immunoprecipitation between endogenous CCT4 and Rheb using adult extracts. Asterisk indicates heavy chain IgG. b Co-immunoprecipitation between endogenous CCT1 and Rheb using adult extracts. c MBP-pulldown assay between MBP-Rheb and GST-CCT4. Rheb?49–63 and Rheb1–48 show little binding to CCT4 compared with the full-length Rheb. Sequence comparison in the related regions of VHL and Rheb is shown in (c'). Mutated Rheb constructs are also shown. d Co-immunoprecipitation between endogenous CCT4 and S6K using adult extracts. Asterisk indicates heavy chain IgG. e Co-immunoprecipitation between endogenous CCT4 and TOR using adult extracts. f Co-immunoprecipitation between endogenous CCT4 and Rheb using S2R+ cell extracts