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. 2017 Mar 24;7:391. doi: 10.1038/s41598-017-00149-0

Figure 3.

Figure 3

mTORC1 activity and RAPTOR levels in ReNcell VM cells are dependent on USP9X. (A) Immunoblot analysis of growth factor induction of mTORC1 and mTORC2 pathways in absence of USP9X. USP9X was depleted from 2193 and 4774 cells by 72 hours exposure to doxycycline before growth factors (EGF/FGF) were removed from ReNcell VM medium 24 hours prior to protein collection (0 minutes) and then 5 and 15 minutes after re-addition of EGF/FGF. Both pS6 and pAkt levels increased upon exposure to EGF/FGF (compare 0 to 5 and 15 min) indicating both mTORC1 and mTORC2 pathway activation, respectively. However, the extent of induction for pS6 was markedly reduced in the absence of USP9X (compare plus and minus Dox at 5 and 15 mins in 2193 and 4774). Total S6 and Total AKT were not affected by USP9X depletion. Representative of three biological repeats. (B) Diminished mTORC1 activation in ReNcell VM cells, as determined by pS6 levels, was evident after only 24 hours doxycycline treatment, consistent with the xCELLigence data. (C) Phospho and Total mTOR levels were unaffected by the absence of USP9X. Representative of two biological replicates. Full length blots presented in Supplementary Fig. S8. (D) RAPTOR protein levels were decreased in ReNcell VM cells with depleted USP9X (2193 and 4774 plus doxycycline) Representative of three biological replicates. (E) Densitometric quantitation, of immunoblots from two biological replicates, of initial pS6 induction compared to total S6 levels 5 mins after addition of EGF/FGF in the absence or presence of doxycycline. The treatment of cells with doxycycline significantly attenuated the level of pS6 in 2193 and 4774 cells only. (F) Densitometric quantitation of immunoblots, from three biological replicates, of RAPTOR protein, relative to β-tubulin control, at time 0 hour in the absence or presence of doxycycline. The treatment of cells with doxycycline significantly attenuated the level of RAPTOR in 2193 and 4774 cells only. All data are shown as the means ± SEM. Statistical significance was assessed by the unpaired Student’s t test. *p < 0.05.