Figure 5. Altered mitochondria metabolism increases ROS and glucose dependence in cells expressing low amounts of TMX1. (A) Quantification of surviving cells upon 24-h glucose deprivation. HeLa cells and TMX1 heterozygous KO HeLa cells were incubated in glucose-free medium (supplemented with galactose) or control condtions (C) and subsequently analyzed for positive Annexin V and propidium iodide (PI) signals. The amount of dead cells was normalized to controls, and results from three independent experiments were graphed. P > 0.05 for all; standard error indicated. (B) Quantification of surviving HeLa cells upon glucose deprivation. Cells were transfected with RNAi against TMX1. After 48 h, cells were incubated in glucose-free medium (supplemented with galactose) and subsequently analyzed for positive Annexin V and PI signals. The amount of dead cells was normalized to the scrambled siRNA controls, and results from three independent experiments were graphed. ***, P < 0.000001; standard error indicated. (C) Quantification of surviving A375P cells upon glucose deprivation. Cells were transfected with RNAi against TMX1, as well as a plasmid encoding for FLAG-tagged TMX1. After 48 h, cells were incubated in glucose-free medium (supplemented with galactose) and subsequently analyzed for positive Annexin V and PI signals. The amount of dead cells was normalized to the scrambled siRNA controls, and results from three independent experiments were graphed. ***, P = 0.001679 for FLAG-TMX1; **, P = 0.01947 for siTMX1; standard error indicated. (D) Measurement of the cellular ROS production via DCF in control HeLa and TMX1 heterozygous KO cells (left). ROS-dependent fluorescence was determined via flow cytometry. Results from three independent experiments are summarized as a graph (*, P = 0.01; standard error expressed vs. HeLa). Measurement of the cellular ROS production via HyPer in control HeLa and A375P cells and cells transfected with FLAG-tagged TMX1 as well as siTMX1 (right). ROS-dependent fluorescence was determined via flow cytometry. Results from three independent experiments are summarized as a graph (**, P = 0.00038 for HeLa and 0.0027 for A375P; standard error expressed vs. HeLa and A375P controls). (E) Growth curve for HeLa WT and heterozygous TMX1 KO cells. Cells were seeded at 100,000 per well, and cells were quantified 24, 48, 72, and 96 h after seeding; standard error indicated. (F) Assessment of glycolytic enzymes. Analysis of HeLa control and TMX1 heterozygous KO and A375P lysates transfected as indicated with FLAG-tagged TMX1 or siTMX1. Lysates were probed for hexokinases I and II and normalized to actin.
Figure 5.

Altered mitochondria metabolism increases ROS and glucose dependence in cells expressing low amounts of TMX1. (A) Quantification of surviving cells upon 24-h glucose deprivation. HeLa cells and TMX1 heterozygous KO HeLa cells were incubated in glucose-free medium (supplemented with galactose) or control condtions (C) and subsequently analyzed for positive Annexin V and propidium iodide (PI) signals. The amount of dead cells was normalized to controls, and results from three independent experiments were graphed. P > 0.05 for all; standard error indicated. (B) Quantification of surviving HeLa cells upon glucose deprivation. Cells were transfected with RNAi against TMX1. After 48 h, cells were incubated in glucose-free medium (supplemented with galactose) and subsequently analyzed for positive Annexin V and PI signals. The amount of dead cells was normalized to the scrambled siRNA controls, and results from three independent experiments were graphed. ***, P < 0.000001; standard error indicated. (C) Quantification of surviving A375P cells upon glucose deprivation. Cells were transfected with RNAi against TMX1, as well as a plasmid encoding for FLAG-tagged TMX1. After 48 h, cells were incubated in glucose-free medium (supplemented with galactose) and subsequently analyzed for positive Annexin V and PI signals. The amount of dead cells was normalized to the scrambled siRNA controls, and results from three independent experiments were graphed. ***, P = 0.001679 for FLAG-TMX1; **, P = 0.01947 for siTMX1; standard error indicated. (D) Measurement of the cellular ROS production via DCF in control HeLa and TMX1 heterozygous KO cells (left). ROS-dependent fluorescence was determined via flow cytometry. Results from three independent experiments are summarized as a graph (*, P = 0.01; standard error expressed vs. HeLa). Measurement of the cellular ROS production via HyPer in control HeLa and A375P cells and cells transfected with FLAG-tagged TMX1 as well as siTMX1 (right). ROS-dependent fluorescence was determined via flow cytometry. Results from three independent experiments are summarized as a graph (**, P = 0.00038 for HeLa and 0.0027 for A375P; standard error expressed vs. HeLa and A375P controls). (E) Growth curve for HeLa WT and heterozygous TMX1 KO cells. Cells were seeded at 100,000 per well, and cells were quantified 24, 48, 72, and 96 h after seeding; standard error indicated. (F) Assessment of glycolytic enzymes. Analysis of HeLa control and TMX1 heterozygous KO and A375P lysates transfected as indicated with FLAG-tagged TMX1 or siTMX1. Lysates were probed for hexokinases I and II and normalized to actin.

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