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Results: 7

1.
FIGURE 7.

FIGURE 7. From: Raptor and Rheb Negatively Regulate Skeletal Myogenesis through Suppression of Insulin Receptor Substrate 1 (IRS1).

mTOR regulates skeletal myogenesis through multiple pathways. See text for details.

Yejing Ge, et al. J Biol Chem. 2011 October 14;286(41):35675-35682.
2.
FIGURE 6.

FIGURE 6. From: Raptor and Rheb Negatively Regulate Skeletal Myogenesis through Suppression of Insulin Receptor Substrate 1 (IRS1).

The function of raptor and Rheb in myogenesis is mediated by IRS1. A, C2C12 myoblasts were transduced overnight with lentiviruses expressing shRNA for IRS1, and subjected to 2-day puromycin selection followed by 3-day differentiation. Differentiated myocytes were lysed for Western analyses. B, myoblasts were co-infected overnight with lentiviruses expressing shRNAs for IRS1 and raptor or Rheb, and treated as in A. Scramble (Scr) was a non-targeting shRNA control. Tubulin served as a loading control. Western bands were quantified and normalized to tubulin control. All data shown are mean ± S.D. (n = 3 for each condition). One-sample t test was performed to compare each data to Scramble control. *, p < 0.05; **, p < 0.01.

Yejing Ge, et al. J Biol Chem. 2011 October 14;286(41):35675-35682.
3.
FIGURE 5.

FIGURE 5. From: Raptor and Rheb Negatively Regulate Skeletal Myogenesis through Suppression of Insulin Receptor Substrate 1 (IRS1).

mTOR/raptor negatively regulates IRS1 protein levels. A, C2C12 myoblasts were treated with 50 nm rapamycin for 30 min (left panels), or infected overnight with mTOR lentivirus followed by 2-day puromycin selection (right panels), before cell lysis and Western analyses. B, myoblasts were transduced overnight with lentiviruses expressing shRNA for raptor or Rheb (left panels), or S6K1 (right panels), followed by 2-day puromycin selection before cell lysis and Western analyses. C, myoblasts were infected overnight with lentiviruses expressing shRNA for raptor or Rheb followed by 2-day puromycin selection; myoblasts (MB) or time-course differentiating myocytes (diff. day 0–3) were lysed for Western analyses. D, 3-day differentiated myocyte were treated with 50 nm rapamycin for 30 min and lyzed for Western analyses. E, 3-day differentiated myocyte depleted with raptor or Rheb as described in C were lysed for Western blot. Scramble (Scr) was a non-targeting shRNA control. Tubulin served as a loading control. Results were repeated at least three times with representative blots shown.

Yejing Ge, et al. J Biol Chem. 2011 October 14;286(41):35675-35682.
4.
FIGURE 2.

FIGURE 2. From: Raptor and Rheb Negatively Regulate Skeletal Myogenesis through Suppression of Insulin Receptor Substrate 1 (IRS1).

Rheb, but not S6K1, negatively regulates myoblast differentiation. C2C12 myoblasts were transduced overnight with lentiviruses expressing shRNAs for Rheb (A–C) or S6K1 (D–F) (Scramble or Scr as a non-targeting control), and subjected to puromycin selection for 2 days followed by differentiation for 3 days. A and D, differentiated myocytes were stained for MHC (green) and DAPI (red). B and E, myocytes were quantified for differentiation index, fusion index, and average myotube size (myonuclei number per myotube). C and F, cells were lysed for Western analyses, and band intensities were quantified by densitometry and normalized to tubulin control. All data shown are mean ± S.D. (n = 3 for each condition). For B & E, paired t test was performed to compare each data to Scramble control. For C and F, one-sample t test was performed to compare each data to Scramble control. *, p < 0.05; **, p < 0.01.

Yejing Ge, et al. J Biol Chem. 2011 October 14;286(41):35675-35682.
5.
FIGURE 1.

FIGURE 1. From: Raptor and Rheb Negatively Regulate Skeletal Myogenesis through Suppression of Insulin Receptor Substrate 1 (IRS1).

mTOR and raptor have opposite roles in myoblast differentiation. C2C12 myoblasts were transduced overnight with lentiviruses expressing shRNAs for mTOR (A–C), raptor (D–F), or rictor (G–I) (Scramble or Scr as a non-targeting control), and subjected to puromycin selection for 2 days followed by differentiation for 3 days. A, D, G, differentiated myocytes were stained for MHC (green) and DAPI (red). B, E, H, myocytes were quantified for differentiation index, fusion index, and average myotube size (myonuclei number per myotube). C, F, I, cells were lysed for Western analyses, and band intensities were quantified by densitometry and normalized to tubulin control. All data shown are mean ± S.D. (n = 3 for each condition). For B, E, H, paired t test was performed to compare each data to Scramble control. For C, F, I, one-sample t test was performed to compare each data to Scramble control. *, p < 0.05; **, p < 0.01.

Yejing Ge, et al. J Biol Chem. 2011 October 14;286(41):35675-35682.
6.
FIGURE 3.

FIGURE 3. From: Raptor and Rheb Negatively Regulate Skeletal Myogenesis through Suppression of Insulin Receptor Substrate 1 (IRS1).

Raptor and Rheb negatively regulate myoblast differentiation. A, C2C12 myoblasts were transfected with HA-raptor or Flag-Rheb together with pCDNA3 (vector), selected with G418 for 2 days, and then induced to differentiate for 3 days. Cells were fixed and stained for MHC (green) and DAPI (red). B, myocytes in A were quantified for differentiation index, fusion index, and average myotube size (myonuclei number per myotube). C, cells were transfected as in A, selected with G418 for 10 days to establish stably transfected pools. Expression of recombinant raptor and Rheb was assessed by Western blotting. D, stably transfected cells were induced to differentiate for 3 days, and lysed for Western blotting at the indicated time points. E, myoblasts were transfected with Myc-S6K1 or Flag-4E-BP1, selected with G418 for 2 days, and then induced to differentiate for 3 days. Differentiated myocytes were lysed for Western blotting. Tubulin served as a loading control. Each experiment was repeated at least three times with representative blots shown or mean ± S.D. For data in B, paired t test was performed to compare each data to vector control. **, p < 0.01.

Yejing Ge, et al. J Biol Chem. 2011 October 14;286(41):35675-35682.
7.
FIGURE 4.

FIGURE 4. From: Raptor and Rheb Negatively Regulate Skeletal Myogenesis through Suppression of Insulin Receptor Substrate 1 (IRS1).

mTOR/raptor negatively regulates Akt activation. C2C12 myoblasts were transduced overnight with lentiviruses expressing shRNAs for raptor or Rheb (A), mTOR (B), or S6K1 (D) (Scr as a non-targeting control), and subjected to puromycin selection for 2 days followed by differentiation for 3 days and lysis for Western blotting. C, cells were transduced with lentiviruses expressing shRNAs for raptor, Rheb or mTOR, and drug-selected as described above, followed by incubation in differentiation medium for 12 h in the presence of 300 ng/ml IGF-II, and then lysis for Western blotting. The scr/no IGF-II lane was from the same Western blot as the +IGF-II lanes for each protein blotted (the separation of lanes in this figure is due to removal of other lanes irrelevant to the experiment), therefore, the band intensities could be directly compared. Band intensities were quantified and normalized to tubulin control. Black bars: pT389-S6K1; gray bars: pS473-Akt; white bars: pT308-Akt. All data shown are mean ± S.D. (n = 3 for each condition). For A, B, and D, one-sample t test was performed to compare each data to Scr control. For C, the “+IGF-II” scramble samples were compared with –IGF-II scramble control by one-sample t test; the other +IGF-II samples were compared with their corresponding +IGF-II scramble samples by paired t test. *, p < 0.05; **, p < 0.01.

Yejing Ge, et al. J Biol Chem. 2011 October 14;286(41):35675-35682.

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