Journal: bioRxiv

Article Title: Depletion of BRCA1 Potentiates Progestin-Induced Cytoskeletal Changes in an Ovarian Cancer Cell Model

doi: 10.64898/2026.01.02.697409

Figure Lengend Snippet: (A) Basal protein expression of ES2 PR-B+ KO-BRCA1 cell models. Top: Western blot for BRCA1, PR. HDAC2 = loading control. Cell line HCC1937 = negative control for BRCA1. Values under blots represent normalized densitometry units (NDU) for the protein of interest/respective loading control. Graph: NDU for BRCA1 (n=3) shown as mean ± SD, with significance shown as ** p ≤ 0.0030, **** p < 0.0001. (B) Basal transcriptional regulation of BRCA1 mRNA. TBP (TATA-box binding protein) = housekeeping gene. Graph: mean ± SD, **** p< 0.0001. (C) Top Blot: Western blot of BRCA1. Bottom Blot: Western blot for PR, and phosphorylated PR at serine sites (Ser294, Ser345, Ser190, & Ser81) following treatment with vehicle (ethanol) or 10nM R5020 for 1 hour. GAPDH = loading control. Values under blots represent NDU for the protein of interest/respective loading control. Graph: NDU for S294 (top) and S81 (bottom). (D) Transcriptional regulation of PR, FOXO1, and p21 mRNA following treatment with vehicle (ethanol) or 10nM R5020 for 6 hours. TBP = housekeeping gene. Graph represents the mean ± SD, **** p < 0.0001.

Article Snippet: ES2 cells were cultured in McCoy’s 5A medium (ATCC, 30-2007) supplemented with 10% charcoal stripped fetal bovine serum (i.e., DCC; Corning, 35072CV), 1% penicillin-streptomycin (i.e., P/S; Gibco, 15070063), and 0.5mg/ml G418 Sulfate (Corning, 61234RG).

Techniques: Expressing, Western Blot, Control, Negative Control, Binding Assay

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

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 silencing or inhibition reduces glycolytic proteins in xenograft ovarian cancer model . (A) In vivo study design of 1 × 10 6 control or DIO3-KD inoculated ES2 cells (B) Total proteins were extracted from representative control or DIO3-KD tumors ( n = 2) and evaluated by WB for HK1, HK2, PFKP, PKM2 and GAPDH. β-actin was used as a loading control. Quantification of normalized bands intensity as fold of control is presented in the right panel. Dashed line represents 100% value of control cells (C) In vivo study design of 1 × 10 6 inoculated ES2 cells treated with vehicle control or 33.3 mg/kG ITYR-DBRMD (D) Total proteins were extracted from representative tumors from control or ITYR-DBRMD treated mice ( n = 2) and evaluated by WB for HK1, HK2, PFKP, PKM2 and GAPDH. β-actin was used as a loading control. Skipping lane is clearly marked by a separating line. Quantification of normalized bands intensity as fold of control is presented in the right panel. Dashed line represents 100% value of control cells. Values are mean ± STE, ∗ p ≤ 0.05; ∗∗ p ≤ 0.005.

Article Snippet: HGSOC ES2 cells were purchased from the ATCC (Cat# CRL-1978).

Techniques: Inhibition, In Vivo, Control

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: Metabolomics analysis of intracellular and extracellular metabolites following DIO3-knockdown in HGSOC cells . Intracellular metabolomics data of triplicates from HGSOC control and DIO3-KD cells analyzed using the MetaboAnalyst tool (A) 2D score plot of Principal component analysis (PCA), left panel. X axis shows principle component 1 that explain 35.9% of the total variance and Y axis shows principle component 2 that explain 23.8% of the total variance. Hierarchical Clustering and heatmap Visualization for the top 50 altered metabolites, right panel (B) Enrichment analysis. Metabolomics results of extracellular metabolites. Four repeats were analyzed by (C) PCA, left panel. X axis explain 58.6% of the total variance and Y axis explain 11.8% of the total variance. Heatmap, right panel (D) Enrichment analysis.

Article Snippet: HGSOC ES2 cells were purchased from the ATCC (Cat# CRL-1978).

Techniques: Knockdown, Control

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: Metabolites alterations in glutamine-related pathways following DIO3 silencing in HGSOC. Panels depict alterations in metabolites related to (A) Intracellular and extracellular glutamine, as well as glutamate secretion. A representative result of glutamate secretion from four independent repeats is presented (B) Krebs cycle. (C) Aspartate metabolism. (D) Urea cycle. (E) Glutathione synthesis and ROS production. Values are mean ± STE, ∗, p ≤ 0.05, ∗∗, p ≤ 0.005, ∗∗∗, p ≤ 0.0005, ∗∗∗∗, p ≤ 0.0001.

Article Snippet: HGSOC ES2 cells were purchased from the ATCC (Cat# CRL-1978).

Techniques:

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: Graphic illustration of metabolites alterations in glutamine-pathways under DIO3 silencing in ovarian cancer . Each panel depicts a major altered pathway related with glutamine upon DIO3 depletion in HGSOC cells: Krebs cycle, Urea cycle and Glutathione synthesis (Created with BioRender.com ). Extracellular alterations are highlighted by pink arrows while intracellular by blue arrows.

Article Snippet: HGSOC ES2 cells were purchased from the ATCC (Cat# CRL-1978).

Techniques:

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 depletion in FT cells alters metabolic pathways that are distinguished from HGSOC. Isolated clones of FT control and DIO3-KD cells were examined for ( A ) DIO3 levels by WB (Upper panel). β-actin was used as loading control. Quantification of normalized bands intensity as fold of control is presented in the right panel. Cells density is presented by microscopy images (Lower panel, 10× objective). ( B ) Cell count as % of control measured by FC. (C) Intracellular levels of T4, measured by LC/MS/MS. (D) Proteomaps using the bionic visualization tool (BionicVis). Metabolism-related pathways are marked in orange and are detailed in the right panel. Alterations in the metabolic proteins FASN and CPT1A are presented in the right panel. (E) A collection of proteins involved in glycolysis and energy production, analyzed by WB. β-tubulin was used as loading control. Quantification of normalized bands intensity as fold of control is presented in the lower panel. Dashed line represents value of 100% (control). WB’s are representative of two independent repeats. (F) FT Control and DIO3-KD cells were seeded (3000cells/well for 96 h) in triplicates and culture media was collected for lactate measurements. A representative result of three independent repeats is presented (G) Venn diagram comparing the proteomics data from DIO3 depletion in FT and HGSOC. Alterations in the proteins TGM2, SLC4A7, NEFL and CPA4 are presented in the right panel. (H) Altered intercellular metabolites detected by metabolomics. Alterations in the metabolites NAA, NAG ATP and FAD are presented in the right panel. Values are mean ± STE, ∗, p ≤ 0.05, ∗∗, p ≤ 0.005.

Article Snippet: HGSOC ES2 cells were purchased from the ATCC (Cat# CRL-1978).

Techniques: Isolation, Clone Assay, Control, Microscopy, Cell Counting, Liquid Chromatography with Mass Spectroscopy

Journal: Genome medicine

Article Title: Pan-cancer analysis identifies CD155 as a promising target for CAR-T cell therapy.

doi: 10.1186/s13073-025-01490-0

Figure Lengend Snippet: Fig. 6 PVRbbz CAR-T cells effectively killed multiple tumor cell lines even at low effector:target ratios and corroborated by cytokines release. A-L Cytotoxicity of PVRbbz CAR-T cells on PC3 (A and G), PANC1 (B and H), U20S (C and I), ES2 (D and J), Huh7 (E and K) and MM.1S (F and L) cells at the indicated effector:target (E:T) ratios after 12 h of co-culturing. Cell Lysis was determined by luciferase and relative IVIS picture was shown. Two independent experiments were performed. M and N The concentrations of TNF-α (M) and IFN-γ (N) in supernatants from cytotoxicity assays at 1:1 effector:target after co-culture with tumor cells for 12 h were measured by ELISA kits. Three independent experiments were performed. Significance was calculated by two-way ANOVA. All error bars represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Article Snippet: Prostate cancer cell lines PC3, C4-2, DU145, multiple myeloma MM.1S cell line, chronic myeloid leukemia K562 cell line, osteosarcoma cell lines U20S and 143B, ovarian cancer cell line ES2 and OVCAR8, pancreatic cancer cell line PANC1 and CAPAN-2, lung cancer cell line H292, colorectal carcinoma cell line HT29 and murine melanoma cell line B16 F10 were obtained from the American Type Culture Collection (ATCC).

Techniques: Lysis, Luciferase, Co-Culture Assay, Enzyme-linked Immunosorbent Assay