Journal: Molecular Metabolism
Article Title: DIO3 depletion attenuates ovarian cancer growth via reduced glycolysis and alterations in glutamine metabolism
doi: 10.1016/j.molmet.2025.102225
Figure Lengend Snippet: DIO3 reduction attenuates glycolysis and elevates OXPHOS in HGSOC cells (A) HGSOC Control and DIO3-KD cells were analyzed by WB for DIO3 (upper panel). Quantification of normalized bands intensity as fold of control is presented in the right panel. All WB’s are representative of at least three independent repeats. Cell density in control and DIO3-KD cells is presented in the lower panel. (10× objective, scale bar: 100 μm) (B) Intracellular levels of the thyroid hormone T4, measured by LC/MS/MS. Values are mean ± STE, ∗, p < 0.05 (C) Proteomaps of the control and DIO3-KD proteomes using the bionic visualization tool. Metabolism-related pathways (orange, right panel). (D) HGSOC Control and DIO3-KD cells were analyzed by WB for a collection of proteins involved in glycolysis and energy production. β-tubulin was used as loading control. Quantification of normalized bands intensity, as fold of control, is presented in the right panel. WB’s are representative of three independent repeats (E) Levels of Fructose-6-phosphate identified by polar metabolites profiling (F) HGSOC control and DIO3-KD cells were seeded (1000cells/well for 96 h) in triplicates and culture media was collected for lactate measurements. A representative result of three independent repeats is presented (G) HGSOC control and DIO3-KD cells were seeded (4x10 4 cells/well) and after 24 h mitochondrial respiration was assessed. OCR was measured under basal conditions followed by sequential injections of oligomycin (1.5 μM), FCCP (1.5 μM) and rotenone (0.5 μM). Bars represent OCR measurements of basal respiration and ATP production. A representative experiment of three independent repeats is shown (H) Levels of ATP/ADP ratio identified by polar metabolites profiling (I) Total proteins were extracted from OVCAR8 control and MCT8 over-expression (MCT8-OE) cells and evaluated by WB for MCT8, DIO3, HK1, PFKP, GAPDH, PKM2 and ATP5A. β-tubulin was used as a loading control. Protein loading for MCT8 is presented in . Quantification of normalized bands intensity as fold of control is presented in the right panel WB’s are representative of three independent repeats (J) Total proteins were extracted from WT and p53-KO ID8 cells and evaluated by WB for p53, DIO3, HK1 and HK2. β-actin and β-tubulin were used as loading control. Quantification of normalized bands intensity as fold of control is presented in the right panel (K) Intracellular levels of the thyroid hormone T3 following 24 h treatment with the DIO3 inhibitor ITYR-DBRMD (250 μM), measured by ELISA. A representative results of at least four independent repeats is shown. Values are mean ± STE ∗∗, p < 0.005 (L) 50 × 10 3 ES2 cells were seeded in 24-well plates and treated daily with ITYR-DBRMD (250 nM, 500 nM, 1 μM, 2.5 μM, 5 μM, 7.5 μM and 10 μM), or vehicle control (DMSO). After 96 h, total proteins were extracted and analyzed by WB for the glycolytic proteins HK1, PFKP, GAPDH and PKM2. β-actin was used as a loading control. Quantification of normalized bands intensity as fold of control is presented in the right panel. Experiments are representative of three independent repeats. Values are mean ± STE, ∗ p ≤ 0.05; ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.0002, ∗∗∗∗ p ≤ 0.0001. (M) 50 × 10 3 OVCAR8 cells were seeded in 24-well plates and treated daily with 1 μM ITYR-DBRMD, or vehicle control (DMSO). After 72 h RNA was extracted and RNA-Seq analysis was performed.
Article Snippet: HGSOC ES2 cells were purchased from the ATCC (Cat# CRL-1978).
Techniques: Control, Liquid Chromatography with Mass Spectroscopy, Over Expression, Enzyme-linked Immunosorbent Assay, RNA Sequencing
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