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. 2022 Mar 7;25(3):103912. doi: 10.1016/j.isci.2022.103912

Figure 6.

Figure 6

IVM reduces TELO2, phosphatidylinositol 3-kinase-related kinase (PIKK) levels and mTOR substrate phosphorylation levels

(A) Short-term IVM treatment reduced TELO2 and β-catenin, but not PIKK levels. HEK293 cells were treated with 5 or 10 μM of IVM for 3, 9, or 24 h in 1% FBS/DMEM. Subsequently, the cell lysates were probed for TELO2, cytoplasmic β-catenin, mTOR, ataxia telangiectasia mutated (ATM), ATM-related and Rad3-related (ATR), DNA-dependent protein kinase (DNA-PK), and actin through western blotting with specific antibodies. The band intensities were quantified, normalized to the actin levels, and indicated as values relative to the control (0 h). Data are presented as the means ± SDs (n = 3 biological replicates). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, one-way ANOVA with Tukey’s test.

(B) Long-term IVM treatment reduced PIKKs. HEK293 cells were treated with 5 μM IVM for 72 h in 1% FBS/DMEM. The cell lysates were probed through western blotting with the indicated antibodies (the left panels). The band intensities were quantified, normalized to the actin levels, and indicated as values relative to the control (DMSO) (the right panels). Data are presented as the means ± SDs (n = 3 biological replicates). ∗p < 0.05, Welch’s t-test.

(C) Short-term IVM treatment reduced AKT and S6 kinase phosphorylation levels. HEK293 cells were treated with 10 μM IVM for 3 h in 1% FBS/DMEM. The cell lysates were probed through western blotting with the indicated antibodies (left panels). The band intensities were quantified, normalized by the total protein levels, and indicated as values relative to the control (DMSO) (the right panels). Data are presented as the means ± SDs (n = 3 biological replicates). ∗∗p < 0.01, Welch’s t-test. See also Figures S6 and S7.

(D) Torin2 reduced the cytoplasmic level of β-catenin. HEK293 cells were treated with 0.1 μM Torin2 or rapamycin for 3 h in 1% FBS/DMEM. The cytoplasmic proteins were probed through western blotting with the indicated antibodies (the left panels). The band intensities were quantified, normalized to the actin levels, and indicated as values relative to the control (DMSO; the right panels). Data are presented as the means ± SDs (n = 3 biological replicates). ∗∗p < 0.01, Welch’s t-test. See also Figure S8.