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