Figure 4.

mTOR signaling activity is elevated in fbxw7 mutant larvae. A, Western blot showing levels of phospho-S6 (pS6) in extracts obtained from wild-type, fbxw7 mutant, mtor mutant, and wild-type and fbxw7 mutants treated with rapamycin. The blot was also probed to detect total S6 levels. B–C′, Representative transverse spinal cord sections of wild-type and fbxw7 mutant larvae carrying the olig2:EGFP transgene and processed for immunohistochemistry to detect phospho-S6. Arrows and arrowheads indicate phospho-S6⁺ and phospho-S6⁻ oligodendrocyte lineage cells, respectively. Outlined boxes show enlarged images of phospho-S6⁺ cells. D–G′′, Representative confocal microscope images of spinal cords of whole larvae used for anti-phospho-S6 quantification, dorsal up. Oligodendrocyte lineage cells, marked by olig2:EGFP expression, are indicated by arrows. Treatment with rapamycin eliminated phospho-S6 labeling in wild-type (F–F′′) and fbxw7 mutant (G–G′′) larvae. G, Graph showing the relative percentages of phospho-S6⁺ and phospho-S6⁻ olig2:EGFP⁺ oligodendrocyte lineage cells. n = 12 wild-type, 5 fbxw7^−/−, 4 rapamycin-treated wild-type and 3 rapamycin-treated fbxw7^−/− larvae. *p = 0.0227; ****p < 0.0001, two-sided Fisher's exact test. H, Scatter plot showing relative fluorescence intensities of anti-phospho-S6 labeling in individual olig2:EGFP⁺ oligodendrocyte lineage cells. n = 39 cells in wild-type, 50 cells in fbxw7^−/− and 8 cells in rapamycin-treated wild-type larvae. ***p < 0.001, two-sided unpaired t test.