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. 2018 Feb 5;217(2):517–526. doi: 10.1083/jcb.201706027

Figure 2.

Figure 2.

dTBC1D7 regulated growth in a cell-nonautonomous manner. (A) Knockdown of TSC1, TSC2, or TSC1 and TSC2, but not dTBC1D7, in S2 cells by RNAi increased the phosphorylation levels of S6K (pS6K). DsRNA for GFP was used as a negative control. The efficiency of TBC1D7 knockdown in S2 cells was ∼60%, as determined by qPCR. S6K mRNA levels were not changed among the groups. (B) Western blot of pS6K in the wild-type and dTBC1D7 mutant flies. S6K mRNA levels were not changed among the groups. (C) Quantification of relative pS6K levels from three biological replicates from panel B. (D–E) Cells mutant for dTBC1D7 were the same sizes as the neighbor cells. (D) Clones of homozygous dTBC1D7KO cells marked with GFP were generated in fat body by the MARCM system, and cell boundaries were stained with phalloidin. Bar, 50 µm. (E) Quantification of the dTBC1D7KO and neighbor cells. At least 100 clones in six individuals were measured. (F–G) Overexpression of dTBC1D7 did not affect cell growth. (F) The dTBC1D7 overexpression (+dTBC1D7) clones were marked with GFP. Bar, 50 µm. (G) The cell size quantification of +dTBC1D7 clones. At least 100 clones in six individuals were measured. Bars, 50 µm. Error bars indicate SD, and significant differences were determined with Student’s t test at indicated genotypes. (H) The dTBC1D7KO fat body cells showed membrane localization of GPH. Bar, 50 µm. (I) Levels of pAKT and pS6K in fat body of wild-type, ilp21, and dTBC1D7KO flies as assessed by using Western blot analysis. ns, not significant.