Figure 3.
Figure 3. Reduction of GSH and increase in ROS in THZ1-treated TSC-deficient cells. (A) Graph of steady-state metabolite levels in 621.101.TSC2− cells in response to THZ1 at 30 nm for 6 h in comparison to vehicle control. The graph shows the log2 fold change for each metabolite. Arrow indicates GSH. n = 3 independent samples. (B) Heat map showing the top 15 metabolites with greatest change in 621.101.TSC2− cells, with comparison to control (CTRL) and similarly treated 621.101.TSC2-intact cells (n = 3 samples). The scale is log twofold-change. (C) Heat map showing the top 25 metabolites with greatest change in HCV.29.TSC1− and MEF.TSC2− cells treated with THZ1 at 30 nm for 6 h (n = 3 independent samples) versus control (n = 3 independent samples). (D) Normalized ROS levels in TSC-deficient and TSC-intact cells treated with control, rapamycin (RAP; 20 nM), THZ1 (30 nM), or the combination for 48 h. Each data point represents the mean ± SEM of three independent experiments. Measured using DCFDA. (E) Normalized ROS levels in TSC-deficient and TSC-intact cells treated with control, THZ1 (30 nM), GSH-MEE (2 mM), or the combination for 48 h. Each data point represents the mean ± SEM of three independent experiments. Measured using DCFDA. (F) Rescue of THZ1-induced cell death by GSH-MEE. HCV.29.TSC1− and HCV.29.TSC1+ cells were treated with DMSO (vehicle), THZ1 (30 nM), GSH-MEE (2 mM), or the combination for 48 h. Phase-contrast images are at top. Cell death (%) is shown at bottom for these treatments, measured by Trypan blue staining. Each data point represents the mean ± SEM of three independent experiments. Scale bar, 100 µm. (G) Confocal microscope images of HCV.29.TSC1− cells showing localization of ROS, by staining with MitoSOX (red, 5 µM) and Mitotracker Green (200 nM) in cells treated with DMSO (vehicle), RAP (20 nM), THZ1 (30 nM), or the combination (two independent experiments performed; ≥10 images per slide were analyzed for each condition. Scale bar, 10 µm. (H) Mitochondrial-specific ROS levels were determined by measuring MitoSOX Red fluorescence in HCV.29.TSC1− and HCV.29.TSC1+ cells treated with control, THZ1 (30 nM), GSH-MEE (2 mM), or the combination for 48 h. Each data point represents the mean ± SEM of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Reduction of GSH and increase in ROS in THZ1-treated TSC-deficient cells. (A) Graph of steady-state metabolite levels in 621.101.TSC2− cells in response to THZ1 at 30 nm for 6 h in comparison to vehicle control. The graph shows the log2 fold change for each metabolite. Arrow indicates GSH. n = 3 independent samples. (B) Heat map showing the top 15 metabolites with greatest change in 621.101.TSC2− cells, with comparison to control (CTRL) and similarly treated 621.101.TSC2-intact cells (n = 3 samples). The scale is log twofold-change. (C) Heat map showing the top 25 metabolites with greatest change in HCV.29.TSC1− and MEF.TSC2− cells treated with THZ1 at 30 nm for 6 h (n = 3 independent samples) versus control (n = 3 independent samples). (D) Normalized ROS levels in TSC-deficient and TSC-intact cells treated with control, rapamycin (RAP; 20 nM), THZ1 (30 nM), or the combination for 48 h. Each data point represents the mean ± SEM of three independent experiments. Measured using DCFDA. (E) Normalized ROS levels in TSC-deficient and TSC-intact cells treated with control, THZ1 (30 nM), GSH-MEE (2 mM), or the combination for 48 h. Each data point represents the mean ± SEM of three independent experiments. Measured using DCFDA. (F) Rescue of THZ1-induced cell death by GSH-MEE. HCV.29.TSC1− and HCV.29.TSC1+ cells were treated with DMSO (vehicle), THZ1 (30 nM), GSH-MEE (2 mM), or the combination for 48 h. Phase-contrast images are at top. Cell death (%) is shown at bottom for these treatments, measured by Trypan blue staining. Each data point represents the mean ± SEM of three independent experiments. Scale bar, 100 µm. (G) Confocal microscope images of HCV.29.TSC1− cells showing localization of ROS, by staining with MitoSOX (red, 5 µM) and Mitotracker Green (200 nM) in cells treated with DMSO (vehicle), RAP (20 nM), THZ1 (30 nM), or the combination (two independent experiments performed; ≥10 images per slide were analyzed for each condition. Scale bar, 10 µm. (H) Mitochondrial-specific ROS levels were determined by measuring MitoSOX Red fluorescence in HCV.29.TSC1− and HCV.29.TSC1+ cells treated with control, THZ1 (30 nM), GSH-MEE (2 mM), or the combination for 48 h. Each data point represents the mean ± SEM of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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