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

Journal: Regenerative Therapy

Article Title: WTAP stabilizes MMP12 expression to promote the malignant phenotypes of esophageal cancer cells

doi: 10.1016/j.reth.2026.101101

Figure Lengend Snippet: MMP12 silencing inhibited M2 macrophage polarization. THP-1 cells were differentiated into M0 macrophages (THP-1 M0) by treatment with 100 ng/mL PMA for 24 h. (A) Flow cytometry was used to quantify the number of CD68-positive cells. Subsequently, KYSE150 cells were co-cultured with the THP-1-derived macrophages using a Transwell system. (B) The mRNA levels of IL-10, Arg-1, and TGF-β were detected by qRT-PCR. (C) Flow cytometry was used to quantify the number of CD206-positive macrophages. (D) Cell migration analysis by transwell migration assay. ∗ P < 0.05, ∗∗ P < 0.01 and ∗∗∗ P < 0.001.

Article Snippet: For immunophenotyping, single-cell suspensions of both THP-1 and THP-1-M0 cells were labeled with an anti-CD68 antibody (E-AB-F1299L, Elabscience, Wuhan, China).

Techniques: Flow Cytometry, Cell Culture, Derivative Assay, Quantitative RT-PCR, Migration, Transwell Migration Assay

Journal: Regenerative Therapy

Article Title: WTAP stabilizes MMP12 expression to promote the malignant phenotypes of esophageal cancer cells

doi: 10.1016/j.reth.2026.101101

Figure Lengend Snippet: WTAP silencing inhibited M2 macrophage polarization by regulating MMP12. THP-1 cells were differentiated into M0 macrophages (THP-1 M0) by treatment with 100 ng/mL PMA for 24 h. KYSE150 cells were transfected with si-WTAP, MMP12 overexpression plasmid, or the matched control (si-NC and oe-NC). Subsequently, these KYSE150 cells were co-cultured with the THP-1-derived macrophages using a Transwell system. (A) The mRNA levels of IL-10, Arg-1, and TGF-β were detected by qRT-PCR. (B) Flow cytometry was used to quantify the number of CD206-positive macrophages. (C) Cell migration analysis by transwell migration assay. ∗ P < 0.05, ∗∗ P < 0.01 and ∗∗∗ P < 0.001.

Article Snippet: For immunophenotyping, single-cell suspensions of both THP-1 and THP-1-M0 cells were labeled with an anti-CD68 antibody (E-AB-F1299L, Elabscience, Wuhan, China).

Techniques: Transfection, Over Expression, Plasmid Preparation, Control, Cell Culture, Derivative Assay, Quantitative RT-PCR, Flow Cytometry, Migration, Transwell Migration Assay

Journal: Regenerative Therapy

Article Title: WTAP stabilizes MMP12 expression to promote the malignant phenotypes of esophageal cancer cells

doi: 10.1016/j.reth.2026.101101

Figure Lengend Snippet: MMP12 silencing inhibited M2 macrophage polarization. THP-1 cells were differentiated into M0 macrophages (THP-1 M0) by treatment with 100 ng/mL PMA for 24 h. (A) Flow cytometry was used to quantify the number of CD68-positive cells. Subsequently, KYSE150 cells were co-cultured with the THP-1-derived macrophages using a Transwell system. (B) The mRNA levels of IL-10, Arg-1, and TGF-β were detected by qRT-PCR. (C) Flow cytometry was used to quantify the number of CD206-positive macrophages. (D) Cell migration analysis by transwell migration assay. ∗ P < 0.05, ∗∗ P < 0.01 and ∗∗∗ P < 0.001.

Article Snippet: EC cell (KYSE-150 and TE-10), normal esophageal epithelial cells (HEEC) and THP-1 cells were provided by Procell (Wuhan, China).

Techniques: Flow Cytometry, Cell Culture, Derivative Assay, Quantitative RT-PCR, Migration, Transwell Migration Assay

Journal: Regenerative Therapy

Article Title: WTAP stabilizes MMP12 expression to promote the malignant phenotypes of esophageal cancer cells

doi: 10.1016/j.reth.2026.101101

Figure Lengend Snippet: WTAP silencing inhibited M2 macrophage polarization by regulating MMP12. THP-1 cells were differentiated into M0 macrophages (THP-1 M0) by treatment with 100 ng/mL PMA for 24 h. KYSE150 cells were transfected with si-WTAP, MMP12 overexpression plasmid, or the matched control (si-NC and oe-NC). Subsequently, these KYSE150 cells were co-cultured with the THP-1-derived macrophages using a Transwell system. (A) The mRNA levels of IL-10, Arg-1, and TGF-β were detected by qRT-PCR. (B) Flow cytometry was used to quantify the number of CD206-positive macrophages. (C) Cell migration analysis by transwell migration assay. ∗ P < 0.05, ∗∗ P < 0.01 and ∗∗∗ P < 0.001.

Article Snippet: EC cell (KYSE-150 and TE-10), normal esophageal epithelial cells (HEEC) and THP-1 cells were provided by Procell (Wuhan, China).

Techniques: Transfection, Over Expression, Plasmid Preparation, Control, Cell Culture, Derivative Assay, Quantitative RT-PCR, Flow Cytometry, Migration, Transwell Migration Assay

Journal: International Journal of Molecular Medicine

Article Title: Triptolide exerts antiviral effects and alleviates influenza A-induced pneumonia by inhibiting the overactivation of absent in melanoma 2 signaling in immune cells

doi: 10.3892/ijmm.2026.5829

Figure Lengend Snippet: H1N1 infection affects cell viability, inflammatory cytokine secretion and interactions between HBEpiCs and THP-1 cells. (A) CCK-8 assay revealed that HBEpiC viability decreased in a concentration-dependent manner following H1N1 infection. (B) ELISA revealed that the levels of IL-1β, IL-6, TNF-α, and IL-8 in HBEpiCs decreased with increasing H1N1 infection. (C) CCK-8 assay indicated that supernatants from H1N1-infected HBEpiC cultures reduced the viability of THP-1 cells in a dose-dependent manner. (D) ELISA results suggested that the levels of inflammatory cytokines (IL-1β, IL-6, TNF-α and IL-8) in THP-1 cells were decreased following exposure to supernatants from H1N1-infected HBEpiC cultures. (E) Cell adhesion assay revealed that the number of THP-1 cells adhering to HBEpiCs increased with increasing H1N1 concentration (scale bar, 10 μ m). Arrow indicates THP-1 cells that remain attached to the surface of HBEpiCs, highlighting the adhesion interaction between the two cell types. (F) Transwell assay suggested that H1N1 infection enhanced the migration capacity of THP-1 cells, with increased migration observed at higher virus concentrations (scale bar, 50 μ m). The data are presented as the mean ± standard deviation; ** P<0.01, *** P<0.001 vs. control. H1N1, influenza A; HBEpiCs, human bronchial epithelial cells; ELISA, enzyme-linked immunosorbent assay; IL, interleukin; TNF-α, tumor necrosis factor-α; Con, control; MOI, multiplicity of infection.

Article Snippet: HBEpiCs were cultured in bronchial epithelial cells complete culture medium (cat. no. ZQ-1322; Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.), while THP-1 cells were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (HyClone; Cytiva) and 1% penicillin-streptomycin (100X; Beijing Solarbio Science & Technology Co., Ltd.).

Techniques: Infection, CCK-8 Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Cell Adhesion Assay, Transwell Assay, Migration, Virus, Standard Deviation, Control

Journal: International Journal of Molecular Medicine

Article Title: Triptolide exerts antiviral effects and alleviates influenza A-induced pneumonia by inhibiting the overactivation of absent in melanoma 2 signaling in immune cells

doi: 10.3892/ijmm.2026.5829

Figure Lengend Snippet: Differential activation of AIM2 signaling in THP-1 cells and HBEpiCs following H1N1 infection. (A) RT-qPCR analysis revealed that H1N1 infection did not alter AIM2 mRNA levels in HBEpiCs but markedly increased AIM2 mRNA levels in THP-1 cells. (B) LDH release assay indicated that H1N1 infection did not affect LDH release in HBEpiCs but markedly increased LDH-1 release in THP-1 cells. (C) Dual-immunofluorescence staining demonstrated the formation of ASC-AIM2 complexes in THP-1 cells post-H1N1 infection (scale bar, 10 μ m). (D) Western blot analysis revealed no significant changes in AIM2, caspase-1, or GSDMD protein levels in HBEpiCs following H1N1 infection but increased expression of AIM2, caspase-1 and GSDMD in THP-1 cells following H1N1 infection (E). (F) Flow cytometry analysis revealed no significant cell death in HBEpiCs following infection, (G) whereas THP-1 cells treated with the supernatant from H1N1-infected HBEpiC cultures presented markedly increased cell death compared with the control. The data are presented as the mean ± standard deviation; * P<0.05, ** P<0.01, *** P<0.001 vs. control. AIM2, absent in melanoma 2; HBEpiCs, human bronchial epithelial cells; H1N1, influenza A; RT-qPCR, reverse transcription-quantitative PCR; GSDMD, gasdermin D; Con, control; MOI, multiplicity of infection.

Article Snippet: HBEpiCs were cultured in bronchial epithelial cells complete culture medium (cat. no. ZQ-1322; Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.), while THP-1 cells were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (HyClone; Cytiva) and 1% penicillin-streptomycin (100X; Beijing Solarbio Science & Technology Co., Ltd.).

Techniques: Activation Assay, Infection, Quantitative RT-PCR, Lactate Dehydrogenase Assay, Immunofluorescence, Staining, Western Blot, Expressing, Flow Cytometry, Control, Standard Deviation, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Medicine

Article Title: Triptolide exerts antiviral effects and alleviates influenza A-induced pneumonia by inhibiting the overactivation of absent in melanoma 2 signaling in immune cells

doi: 10.3892/ijmm.2026.5829

Figure Lengend Snippet: TP modulates the inflammatory response and immune cell activity in H1N1-infected HBEpiCs and THP-1 cells. (A) No significant changes were observed in HBEpiCs treated with various concentrations of TP (5, 10 and 20 nM) following H1N1 infection compared with the control. (B) After TP treatment, the levels of the inflammatory cytokines IL-1β, IL-6, TNF-α and IL-8 in HBEpiCs were markedly lower than those in the untreated group. (C) The viability of THP-1 cells pretreated with H1N1-infected HBEpiC culture supernatant decreased after TP treatment. (D) The levels of IL-1β, IL-6, TNF-α, and IL-8 in THP-1 cells were markedly lower after TP treatment. (E) The adhesion of THP-1 cells to HBEpiCs induced by H1N1 infection decreased in a dose-dependent manner with increasing TP concentration (scale bar, 10 μ m). Arrow indicates THP-1 cells that remain attached to the surface of HBEpiCs, highlighting the adhesion interaction between the two cell types. (F) The migration capacity of THP-1 cells was markedly reduced when the supernatant from H1N1-infected HBEpiC cultures was treated with TP (scale bar, 50 μ m). The data are presented as the mean ± standard deviation; * P<0.05, ** P<0.01, *** P<0.001 vs. control. TP, triptolide; H1N1, influenza A; HBEpiCs, human bronchial epithelial cells; IL, interleukin; TNF-α, tumor necrosis factor-α; Con, control.

Article Snippet: HBEpiCs were cultured in bronchial epithelial cells complete culture medium (cat. no. ZQ-1322; Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.), while THP-1 cells were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (HyClone; Cytiva) and 1% penicillin-streptomycin (100X; Beijing Solarbio Science & Technology Co., Ltd.).

Techniques: Activity Assay, Infection, Control, Concentration Assay, Migration, Standard Deviation

Journal: International Journal of Molecular Medicine

Article Title: Triptolide exerts antiviral effects and alleviates influenza A-induced pneumonia by inhibiting the overactivation of absent in melanoma 2 signaling in immune cells

doi: 10.3892/ijmm.2026.5829

Figure Lengend Snippet: Effects of TP on AIM2 signaling, LDH leakage, and pyroptosis in HBEpiCs and THP-1 cells. (A) RT-qPCR analysis revealed that TP treatment did not alter AIM2 mRNA expression in HBEpiCs but markedly reduced AIM2 mRNA levels in THP-1 cells. (B) TP did not affect the LDH leakage rate in HBEpiCs but markedly reduced LDH leakage in THP-1 cells. (C) Dual-fluorescence staining revealed that TP treatment decreased the formation of ASC-AIM2 complexes in THP-1 cells (scale bar, 10 μ m). (D) Western blot analysis indicated that TP treatment did not markedly affect the expression of these proteins in HBEpiCs. (E) TP treatment reduced the protein expression of AIM2, caspase-1, and GSDMD in THP-1 cells. (F) Flow cytometry revealed no difference in cell death between HBEpiCs treated with 20 nM TP and the control. (G) In THP-1 cells infected with H1N1 and treated with 20 nM TP, a significant reduction in cell death was identified compared with that in the H1N1 infection group. The data are presented as the mean ± standard deviation; * P<0.05, ** P<0.01, *** P<0.001 vs. control. TP, triptolide; AIM2, absent in melanoma 2; LDH, lactate dehydrogenase; HBEpiCs, human bronchial epithelial cells; RT-qPCR, reverse transcription-quantitative PCR; H1N1, influenza A; GSDMD, gasdermin D; Con, control.

Article Snippet: HBEpiCs were cultured in bronchial epithelial cells complete culture medium (cat. no. ZQ-1322; Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.), while THP-1 cells were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (HyClone; Cytiva) and 1% penicillin-streptomycin (100X; Beijing Solarbio Science & Technology Co., Ltd.).

Techniques: Quantitative RT-PCR, Expressing, Fluorescence, Staining, Western Blot, Flow Cytometry, Control, Infection, Standard Deviation, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Medicine

Article Title: Triptolide exerts antiviral effects and alleviates influenza A-induced pneumonia by inhibiting the overactivation of absent in melanoma 2 signaling in immune cells

doi: 10.3892/ijmm.2026.5829

Figure Lengend Snippet: AIM2 overexpression reverses the immunosuppressive effects of TP in THP-1 cells. (A) Transfection with Ad-AIM2 upregulated the expression of AIM2 compared with that of Ad-NC in THP-1 cells. (B) AIM2 protein levels were elevated in THP-1 cells overexpressing AIM2 compared with control THP-1 cells. (C) In THP-1 cells, AIM2 overexpression increased the LDH leakage rate. (D) AIM2 overexpression reversed the TP-induced reduction in adhesion between THP-1 cells and HBEpiCs (scale bar, 10 μ m). (E) AIM2 overexpression enhanced the migration ability of THP-1 cells compared with that of H1N1-treated control cells and reversed the TP-induced decrease in THP-1 cell migration (scale bar, 50 μ m). (F) AIM2 overexpression reversed the TP-induced reduction in inflammatory cytokine levels. The data are presented as the mean ± standard deviation; * P<0.05, ** P<0.01, *** P<0.001 vs. Con; # P<0.05, ## P<0.01, ### P<0.001 vs. Ad-AIM2. AIM2, absent in melanoma 2; TP, triptolide; LDH, lactate dehydrogenase; HBEpiCs, human bronchial epithelial cells; H1N1, influenza A; GSDMD, gasdermin D; IL, interleukin; TNF-α, tumor necrosis factor-α; Con, control.

Article Snippet: HBEpiCs were cultured in bronchial epithelial cells complete culture medium (cat. no. ZQ-1322; Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.), while THP-1 cells were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (HyClone; Cytiva) and 1% penicillin-streptomycin (100X; Beijing Solarbio Science & Technology Co., Ltd.).

Techniques: Over Expression, Transfection, Expressing, Control, Migration, Standard Deviation

Journal: Regenerative Therapy

Article Title: Lung cancer cell-derived exosomal EHF drives M2 macrophage polarization via transcriptional activation of RNF41 to promote tumor progression

doi: 10.1016/j.reth.2026.101104

Figure Lengend Snippet: EHF promotes lung cancer cell malignancy and M2 macrophage polarization. A549 and H520 cells were transfected with control (Ctrl) or EHF-knockdown (KD-EHF) plasmids. (A-B) Western blot and quantification of EHF protein levels in A549 and H520 cells. (C-D) EdU staining and quantification of EdU + cells in A549 and H520 cells, assessing proliferation. (E-H) Glucose uptake and lactate production assays were conducted using commercial kits to evaluate glycolytic output. (I-J) Flow cytometric analysis of apoptosis. (K-L) Transwell assay was employed to examine cell migration. (M − P) THP-1 cells were differentiated into THP-1-M0 macrophages with 100 ng/mL PMA for 24 h. Culture supernatants from Ctrl- or KD-EHF–transfected A549 and H520 cells were collected and co-incubated with THP-1-M0 macrophages. (M − N) Flow cytometric analysis was performed to assess the proportion of CD206 + M2-polarized macrophages. (O–P) ELISA was conducted to quantify the secretion of M2-associated cytokines (TGF-β1, IL-10, and VEGFA) in the supernatants of THP-1-M0 macrophages. Data are presented as mean ± SD. P < 0.05 denotes statistical significance.

Article Snippet: Human monocyte cell line (THP-1) (Catalog #: YC-D011) were purchased from Guangzhou Ubigene Biosciences Co., Ltd. (Guangzhou, China) and cultured in RPMI-1640 (Gibco) containing 10% FBS (Gibco).

Techniques: Transfection, Control, Knockdown, Western Blot, Staining, Transwell Assay, Migration, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Regenerative Therapy

Article Title: Lung cancer cell-derived exosomal EHF drives M2 macrophage polarization via transcriptional activation of RNF41 to promote tumor progression

doi: 10.1016/j.reth.2026.101104

Figure Lengend Snippet: Exosomes derived from lung cancer cells are internalized by THP-1-M0 macrophages and mediate EHF delivery. Exosomes were isolated from A549 and H520 cells. (A) TEM images showed the typical cup-shaped morphology of exosomes isolated from A549 and H520 cell supernatants (scale bar: 100 nm). (B) NTA of exosomes from A549 and H520 cells. (C) Western blot analysis of exosome markers (CD9 and TSG101) and the endoplasmic reticulum marker Calnexin in exosome lysates and parental A549/H520 cell lysates. (D-E) THP-1-M0 macrophages (differentiated from THP-1 cells with 100 ng/mL PMA for 24 h) were co-incubated with DiI-labeled exosomes isolated from A549 and H520 cells. (D) Fluorescence microscopy images of THP-1-M0 macrophages after co-incubation with PKH67-labeled exosomes (scale bar: 20 μm). (E-F) Western blot and quantification of EHF protein levels in THP-1-M0 macrophages after incubation with exosomes from A549 or H520 cells. Data are presented as mean ± SD. P < 0.05 denotes statistical significance.

Article Snippet: Human monocyte cell line (THP-1) (Catalog #: YC-D011) were purchased from Guangzhou Ubigene Biosciences Co., Ltd. (Guangzhou, China) and cultured in RPMI-1640 (Gibco) containing 10% FBS (Gibco).

Techniques: Derivative Assay, Isolation, Western Blot, Marker, Incubation, Labeling, Fluorescence, Microscopy

Journal: Regenerative Therapy

Article Title: Lung cancer cell-derived exosomal EHF drives M2 macrophage polarization via transcriptional activation of RNF41 to promote tumor progression

doi: 10.1016/j.reth.2026.101104

Figure Lengend Snippet: Exosomal EHF reprograms THP-1-M0 macrophages to promote lung cancer cell malignancy and angiogenesis via paracrine signaling. (A) Schematic of the paracrine co-culture system: Exosomes from control (Ctrl) or EHF-knockdown (KD-EHF) A549/H520 cells were co-incubated with THP-1-M0 macrophages. The medium from these macrophages was used to treat A549/H520 cells or HUVECs. (B-E) THP-1-M0 macrophages were treated with three conditions: blank control (no exosome), exosomes from control-transfected A549/H520 cells (Exo Ctrl ), or exosomes from EHF-knockdown A549/H520 cells (Exo KD−EHF ). (B–C) Flow cytometry analysis of CD206 + M2 macrophage proportions in THP-1-M0 cells. (D-E) ELISA measurement of M2-associated cytokines (TGF-β1, IL-10, and VEGFA). (F-M) The medium from exosome treated macrophages was then used to incubate A549/H520 cells or HUVECs. (F-G) EdU staining and quantification of EdU + proliferating A549 and H520 cells. (H–I) Flow cytometric analysis of apoptosis in A549 and H520 cells. (J-K) Transwell migration assays and quantification of migrated A549 and H520 cells. (L-M) Tube formation assays and quantification of tube numbers in HUVECs. Data are presented as mean ± SD. P < 0.05 denotes statistical significance.

Article Snippet: Human monocyte cell line (THP-1) (Catalog #: YC-D011) were purchased from Guangzhou Ubigene Biosciences Co., Ltd. (Guangzhou, China) and cultured in RPMI-1640 (Gibco) containing 10% FBS (Gibco).

Techniques: Co-Culture Assay, Control, Knockdown, Incubation, Transfection, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Staining, Migration

Journal: Regenerative Therapy

Article Title: Lung cancer cell-derived exosomal EHF drives M2 macrophage polarization via transcriptional activation of RNF41 to promote tumor progression

doi: 10.1016/j.reth.2026.101104

Figure Lengend Snippet: EHF transcriptionally activates RNF41 and mediates its intercellular transfer via exosomes. (A) JASPAR database prediction of the EHF-binding motif within the RNF41 promoter region, localized to positions −828/-821. (B) ChIP-qPCR analysis in THP-1-M0 macrophages. (C) Dual-luciferase reporter assays in 293T cells. (D) WB analysis of EHF protein levels in THP-1-M0 macrophages with EHF knockdown (KD-EHF) or control (Ctrl). (E-F) Western blot and qRT-PCR analysis of RNF41 protein and mRNA levels in THP-1-M0 macrophages with KD-EHF or Ctrl. (G-H) THP-1-M0 macrophages were treated with culture medium (Blank), control exosomes (Ctrl Exo, from A549/H520 cells), or EHF-knockdown exosomes (KD-EHF Exo, from A549/H520-KD-EHF cells). RNF41 protein levels were detected by Western blot. Data are presented as mean ± SD. P < 0.05 denotes statistical significance.

Article Snippet: Human monocyte cell line (THP-1) (Catalog #: YC-D011) were purchased from Guangzhou Ubigene Biosciences Co., Ltd. (Guangzhou, China) and cultured in RPMI-1640 (Gibco) containing 10% FBS (Gibco).

Techniques: Binding Assay, ChIP-qPCR, Luciferase, Knockdown, Control, Western Blot, Quantitative RT-PCR

Journal: Regenerative Therapy

Article Title: Lung cancer cell-derived exosomal EHF drives M2 macrophage polarization via transcriptional activation of RNF41 to promote tumor progression

doi: 10.1016/j.reth.2026.101104

Figure Lengend Snippet: RNF41 mediates exosomal EHF-driven pro-tumorigenic effects in lung cancer. (A) Schematic of the rescue co-culture system: THP-1-M0 macrophages (control or RNF41-overexpressing) were incubated with exosomes from control (Exo Ctrl ) or EHF-knockdown (Exo KD−EHF ) A549/H520 cells. The medium from these co-cultures was used to treat A549/H520 cells or HUVECs. (B–C) Flow cytometry was used to analyze the proportion of CD206 + M2-polarized macrophages in THP-1-M0 cells. (D-E) ELISA was employed to quantify the levels of M2-associated cytokines (TGF-β1, IL-10, and VEGFA) in the CM from macrophages. (F-G) EdU assays were carried out to measure the proliferation of A549 and H520 cells. (H–I) Annexin V/PI flow cytometry was used to assess the apoptosis of A549 and H520 cells treated with CM. (J-K) Transwell assays were performed to evaluate the migration of A549 and H520 cells. (L-M) Tube formation assays were conducted on HUVECs treated with CM. Data are presented as mean ± SD. P < 0.05 denotes statistical significance.

Article Snippet: Human monocyte cell line (THP-1) (Catalog #: YC-D011) were purchased from Guangzhou Ubigene Biosciences Co., Ltd. (Guangzhou, China) and cultured in RPMI-1640 (Gibco) containing 10% FBS (Gibco).

Techniques: Co-Culture Assay, Control, Incubation, Knockdown, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Migration