Journal: Genes & Diseases

Article Title: Suppression of UCP2 alleviates leukemogenesis by enhancing branched-chain amino acids-induced oxidative stress via activating the PI3K/AKT/mTOR signaling pathway

doi: 10.1016/j.gendis.2025.101794

Figure Lengend Snippet: Silencing UCP2 inhibits leukemogenesis and disrupts mitochondrial homeostasis. THP-1 or KG-1 cells were transduced either with lentiviral encoding a scramble control, shUCP2#2, or shUCP2#3 plasmid for 72 h. GFP-positive cells were sorted using flow cytometry. Stable transfectants were sub-cultured before being subjected to (A) cell viability and (B) cell cycle determination by flow cytometry. (C) Cell number was counted using cell countess and normalized to the scramble control. Oxidative stress indexes, including (D) ATP, (E) NADP/NADPH ratio, and (F) GSH/GSSG ratio, were determined according to the manufacturer’s instructions. (G) Mitochondrial ROS, (H) mitochondrial mass, and (I) mitochondrial membrane potential were determined according to the instruction manual. (J) Mitochondrial morphology (indicated by red box) was determined using high-power electron microscopy, the number of mitochondrial fusions was quantified using Image J software, represented as mitochondria number/mm, and data were normalized to the scramble control. Mitochondria morphology was indicated in the red box. UCP2 silencing THP-1 AML xenografts were generated as described, and mice were sacrificed 12 days post-transplantation. Tumorigenesis-related indexes, including (K) mouse body weight, (L) survival, (M) cell viability, (N) the size of spleen and liver, (O) AML engraftment (hCD45 + ), and (P) mitochondrial ROS from bone marrow mononuclear cells, were determined using flow cytometry. All experiments were repeated three times. Data represent mean ± standard deviation from technical triplicates (∗ p < 0.01, ∗∗ p < 0.05, and ∗∗∗ p < 0.001; two-way ANOVA for scramble control versus shUCP2s). UCP2, uncoupling protein 2; AML, acute myeloid leukemia; NADP, nicotinamide adenine dinucleotide phosphate; NADPH, reduced NADP; GSH, reduced glutathione; GSSG, oxidized glutathione; ROS, reactive oxygen species.

Article Snippet: Kasumi-1 (CLR-2724), KG-1 (CRL-8031), and THP-1 (TIB-202) were purchased from ATCC, and MOLM-13 (ACC-554) was purchased from DSMZ-German Collection of Microorganisms and Cell Cultures GmbH.

Techniques: Control, Plasmid Preparation, Flow Cytometry, Cell Culture, Membrane, Electron Microscopy, Software, Generated, Transplantation Assay, Standard Deviation

Journal: Genes & Diseases

Article Title: Suppression of UCP2 alleviates leukemogenesis by enhancing branched-chain amino acids-induced oxidative stress via activating the PI3K/AKT/mTOR signaling pathway

doi: 10.1016/j.gendis.2025.101794

Figure Lengend Snippet: Genetically knockdown of UCP2 up-regulated BCAA in acute myeloid leukemia. THP-1 or KG-1 cells overexpressing either a scramble control, shUCP2#2, or shUCP2#3 stable transfectants were subjected to RNA-sequencing analysis. (A) KEGG pathway analysis of the top signaling pathways that were aberrantly regulated in the above stable transfectants (∗common signaling pathways in both cell lines). THP-1 or KG-1 cells overexpressing either a scramble control, shUCP2#2, or shUCP2#3 stable transfectants were subjected to metabolic mass spectrometry. (B) Heatmap of the expression profile of amino acid metabolism-related gene expression. (C – E) The peak area for the fold chain of amino acid gene expression (data were normalized to scramble control). (F, G) The BCAA content of THP-1 or KG-1 cells overexpressing either a scramble control, shUCP2#2, or shUCP2#3 stable transfectants (data were normalized to the scramble control). All experiments were repeated three times. Data represent mean ± standard deviation from technical triplicates (∗ p < 0.01, ∗∗ p < 0.05, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.005; two-way ANOVA for scramble control versus shUCP2s). BCAA, branched-chain amino acid; UCP2, uncoupling protein 2.

Article Snippet: Kasumi-1 (CLR-2724), KG-1 (CRL-8031), and THP-1 (TIB-202) were purchased from ATCC, and MOLM-13 (ACC-554) was purchased from DSMZ-German Collection of Microorganisms and Cell Cultures GmbH.

Techniques: Knockdown, Control, RNA Sequencing, Protein-Protein interactions, Mass Spectrometry, Expressing, Gene Expression, Standard Deviation

Journal: Genes & Diseases

Article Title: Suppression of UCP2 alleviates leukemogenesis by enhancing branched-chain amino acids-induced oxidative stress via activating the PI3K/AKT/mTOR signaling pathway

doi: 10.1016/j.gendis.2025.101794

Figure Lengend Snippet: BCAA accumulation-induced oxidative stress activates the PI3K/AKT/mTOR signaling pathway in acute myeloid leukemia. THP-1 and KG-1 cells, two acute myeloid leukemia cell lines that exhibits high level of endogenous UCP2, were treated with increasing doses of BCAA (4 μM, 6 μM, and 10 μM) for 24 h before determination of (A) NADP/NADPH ratio, (B) GSH/GSSG ratio, (C) mitochondrial ROS, (D) mitochondrial mass, and (E) mitochondrial membrane potential. (F) Immunoblotting was performed to determine the expression of PI3K/AKT/mTOR signaling proteins. All experiments were repeated three times. Data represent mean ± standard deviation from technical triplicates (∗ p < 0.01, ∗∗ p < 0.05, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.005; two-way ANOVA for the untreated versus the treated. NADP, nicotinamide adenine dinucleotide phosphate; NADPH, reduced NADP; GSH, reduced glutathione; GSSG, oxidized glutathione; ROS, reactive oxygen species; BCAA, branched-chain amino acid; UCP2, uncoupling protein 2.

Article Snippet: Kasumi-1 (CLR-2724), KG-1 (CRL-8031), and THP-1 (TIB-202) were purchased from ATCC, and MOLM-13 (ACC-554) was purchased from DSMZ-German Collection of Microorganisms and Cell Cultures GmbH.

Techniques: Membrane, Western Blot, Expressing, Standard Deviation

Journal: Genes & Diseases

Article Title: Suppression of UCP2 alleviates leukemogenesis by enhancing branched-chain amino acids-induced oxidative stress via activating the PI3K/AKT/mTOR signaling pathway

doi: 10.1016/j.gendis.2025.101794

Figure Lengend Snippet: Lack of BCAA restored UCP2 silencing-induced anti-leukemogenesis and oxidative stress phenotypes. THP-1 or KG-1 cells overexpressing either a scramble control, shUCP2#2, or shUCP2#3 stable transfectants in the proliferating phase were sub-cultured either in a normal or low level of BCAA as described previously before being subject to the following experiments. (A) Cell viability or metabolic-related assays, including (B) mitochondrial ROS, (C) mitochondrial mass, and (D) mitochondrial membrane potential, were determined using flow cytometry. (E) BCAA levels were measured according to the instruction manual. All data were represented as fold change (normalized to shCtrl), and all experiments were repeated three times. Data represent mean ± standard deviation from technical triplicates (∗ p < 0.01, ∗∗ p < 0.05, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.005; two-way ANOVA for normal BCAA versus lack of BCAA). ROS, reactive oxygen species; BCAA, branched-chain amino acid; UCP2, uncoupling protein 2.

Article Snippet: Kasumi-1 (CLR-2724), KG-1 (CRL-8031), and THP-1 (TIB-202) were purchased from ATCC, and MOLM-13 (ACC-554) was purchased from DSMZ-German Collection of Microorganisms and Cell Cultures GmbH.

Techniques: Control, Cell Culture, Membrane, Flow Cytometry, Standard Deviation

Journal: Genes & Diseases

Article Title: Suppression of UCP2 alleviates leukemogenesis by enhancing branched-chain amino acids-induced oxidative stress via activating the PI3K/AKT/mTOR signaling pathway

doi: 10.1016/j.gendis.2025.101794

Figure Lengend Snippet: Genipin induced leukemic cell death via accumulating BCAA. KG-1, THP-1, and MOLM-13 cells were treated either with or without genipin at the indicated doses (20 μM, 40 μM, 60 μM, 80 μM, and 100 μM) for 48 h before determination of (A) cell growth using cell countess, (B) cell viability using flow cytometry, and (C) cell cytotoxicity using CCK8 assay. Data were normalized to the scramble control. UCP2 protein level and mRNA level were determined either using (D) immunoblotting or (E) quantitative PCR upon genipin treatment. Metabolism-related assays, including (F) mitochondrial ROS, (G) mitochondrial mass, and (H) mitochondrial membrane potential, were determined using flow cytometry. Five de novo acute myeloid leukemia primary cells were sub-cultured either in the normal or high level of BCAA culture medium before being treated either with 40 μM or 60 μM genipin for 48 h, respectively. Primary cells were then subjected to (I) cell viability or (J – L) mitochondria-related assays (data were normalized to the vehicle control). All experiments were repeated three times. Data represent mean ± standard deviation from technical triplicates (∗ p < 0.01; ∗∗ p < 0.05; ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.005; two-way ANOVA for the untreated versus the treated, and normal BCAA versus high BCAA). ROS, reactive oxygen species; BCAA, branched-chain amino acid; UCP2, uncoupling protein 2.

Article Snippet: Kasumi-1 (CLR-2724), KG-1 (CRL-8031), and THP-1 (TIB-202) were purchased from ATCC, and MOLM-13 (ACC-554) was purchased from DSMZ-German Collection of Microorganisms and Cell Cultures GmbH.

Techniques: Flow Cytometry, CCK-8 Assay, Control, Western Blot, Real-time Polymerase Chain Reaction, Membrane, Cell Culture, Standard Deviation

Journal: Genes & Diseases

Article Title: Suppression of UCP2 alleviates leukemogenesis by enhancing branched-chain amino acids-induced oxidative stress via activating the PI3K/AKT/mTOR signaling pathway

doi: 10.1016/j.gendis.2025.101794

Figure Lengend Snippet: Supplementation of BCAA enhanced the anti-tumor activity of genipin. Two million THP-1 or MOLM-13 cells were transplanted into six-week-old nude mice via the tail vein injection; meanwhile, mice were administered either PBS (vehicle Ctrl) or 25 mg/kg genipin every two days via intraperitoneal injection. Mice were sacrificed, and tumorigenesis-related indexes, including (A) percentage of AML engraftment, (B) cell viability, and (C) mitochondrial ROS, were assessed. (D) Mice were treated with either PBS (vehicle Ctrl) or 25 mg/kg genipin as described previously. In the high BCAA group, mice were fed with water containing 15 mg/mL valine, 15 mg/mL leucine, and 15 mg/mL isoleucine throughout the entire experiment. In the normal BCAA group, mice were fed with normal sterile ddH 2 O. Mice were sacrificed 12 days post-transplantation. Tumorigenesis-related indexes, including (E, F) mice survival, (G, H) percentage of AML engraftment in bone marrow (BM) and peripheral blood (PBL), (I) cell viability, and (J) mitochondrial ROS, were assessed. (K) The BCAA level was also determined upon withdrawing mouse blood via the posterior ophthalmic venous plexus. The survival analysis was performed using the log-rank (Mantel–Cox) test. All experiments were repeated three times. Data represent mean ± standard deviation from technical triplicates (∗ p < 0.01, ∗∗ p < 0.05, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.005; two-way ANOVA for genipin plus normal BCAA versus genipin plus high BCAA. AML, acute myeloid leukemia; ROS, reactive oxygen species; BCAA, branched-chain amino acid.

Article Snippet: Kasumi-1 (CLR-2724), KG-1 (CRL-8031), and THP-1 (TIB-202) were purchased from ATCC, and MOLM-13 (ACC-554) was purchased from DSMZ-German Collection of Microorganisms and Cell Cultures GmbH.

Techniques: Activity Assay, Injection, Sterility, Transplantation Assay, Standard Deviation