Journal: Oncology Letters

Article Title: Lung squamous cell carcinoma downregulates haptoglobin expression to inhibit M2 macrophage ferroptosis via the hemoglobin-dependent CD163/HO-1 pathway

doi: 10.3892/ol.2026.15558

Figure Lengend Snippet: CM from LUSC cells overexpressing HP leads to ferroptosis in M2 macrophages. (A) THP-1 cells were differentiated into macrophages by treating them with 150 nM PMA for 24 h, followed by culture in fresh RPMI-1640 for 24 h. The macrophages were polarized into M2 macrophages by incubating with IL-4 (20 ng/ml) and IL-13 (20 ng/ml) for 24 h. Flow cytometry was then performed to identify CD163 + CD206 + M2 macrophages. (B) M2 macrophages were cultured with the CM from NCI-H226 and NCI-H520 cells infected with LV- HP or LV-Ctrl in the presence or absence of Hb for 24 h. LDH release was analyzed by ELISA. Treated M2 macrophages were treated as in (B) and flow cytometry was used to detect the proportion of (C) 7-AAD positive cells, as well as the mean fluorescence intensity of (D) FerroOrange and (E) C11-BODIPY. (F) Treated M2 macrophages were observed using transmission electron microscopy. Scale bar, 5 µm. Data are presented as the mean ± SD from five independent experiments and were analyzed using an unpaired t-test or a two-way ANOVA followed by a Tukey's post-hoc multiple comparison analysis. ****P<0.0001. ns, not significant; CM, conditioned media; LUSC, lung squamous cell carcinoma; LDH, lactate dehydrogenase; 7-AAD, 7-aminoactinomycin D; HP, haptoglobin.

Article Snippet: To identify the induction of M2 macrophages, THP-1 cells were treated with 150 nM Phorbol 12-Myristate 13-Acetate (cat. no. HY-18739; MedChemExpress) for 24 h, cultured in fresh RPMI-1640 for 24 h, and then stimulated with IL-4 (20 ng/ml; cat. no. 200-04-5UG; Thermo Fisher Scientific, Inc.) and IL-13 (20 ng/ml; cat. no. 200-13-10UG; Thermo Fisher Scientific, Inc.) for an additional 24 h, all at 37°C.

Techniques: Flow Cytometry, Cell Culture, Infection, Enzyme-linked Immunosorbent Assay, Fluorescence, Transmission Assay, Electron Microscopy, Comparison

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: 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: The Journal of Experimental Medicine

Article Title: DNA-PK interacts with cyclic dinucleotides and inhibits type I interferon responses

doi: 10.1084/jem.20251796

Figure Lengend Snippet: 2′3′-cGAMP interacts with the catalytic pocket of DNA-PKcs. (A) Experimental scheme for B and C. FLAG-tagged DNA-PKcs (F-DNA-PKcs or FLAG-DNA-PKcs) expressed in 293T cells was FLAG was subjected to immunoprecipitation (IP), prior to incubation with 2′3'-cGAMP, release of bound 2′3′-cGAMP, and detection by ELISA. (B) WB analysis of input and FLAG-IP performed as in A was conducted using the indicated antibodies. Representative WB of three independent experiments. (C) 2′3′-cGAMP was measured by ELISA on experiment performed as in A. Graph presents the mean ± SEM of three independent experiments. Statistical significance was calculated by two-tailed Student's t test. (D) Experimental scheme for E. Recombinant DNA-PKcs was immunoprecipitated using a DNA-PKcs–specific antibody, prior to incubation with 2′3′-cGAMP, release of bound 2′3′-cGAMP, and detection by ELISA. (E) Graph represents mean (±SEM) 2′3′-cGAMP levels as measured in mock IgG and DNA-PK–specific IP performed as in D. Statistical significance was calculated by two-tailed Student's t test. n = 3 independent experiments. (F) Experimental scheme for G. FLAG-tagged DNA-PKcs (FLAG-DNA-PKcs) expressed in 293T cells was FLAG purified prior to incubation with biotin or biotinylated 2′3′-cGAMP (C3-2′3′-cGAMP), followed by streptavidin pull-down and WB analysis. (G) WB analysis of input and streptavidin pull-down experiment performed as in F was conducted using a FLAG-specific antibody. Representative WB of three independent experiments. (H) DNA-PKcs (red) and 2′3′-cGAMP (green) subcellular localization was assessed 6 h after iFluor488-2′3′-cGAMP transfection in T98G cells. Immunofluorescence was performed using a DNA-PKcs–specific antibody and DAPI nuclear staining. Representative images of 15–20 images. Scale bars, 5 µm. (I) Quantification of cytosolic DNA-PKcs and iFluor488-2′3′-cGAMP foci colocalization following transfection of T98G cells with mock or fluorescent 2′3′-cGAMP using the CellProfiler software. n = 424 and 558. Statistical significance was calculated by two-tailed Student's t test. (J) Experimental scheme for K. THP-1 cells were processed for TSA in the presence or absence of 2′3′-cGAMP. (K) WB analysis of TSA, as described in J, was conducted using indicated antibodies. Representative WB of three independent experiments. (L) Experimental scheme for M. Purified FLAG-DNA-PKcs was used as input material for TSA in the presence or absence of 2′3′-cGAMP. (M) WB analysis of TSA, performed as in L, was conducted using anti-FLAG antibody. Representative WB of three independent experiments. (N) Representation of the molecular modelling of 2′3′-cGAMP in interaction with DNA-PKcs. (O) ATP hydrolysis by DNA-PK was measured in vitro in presence of NU7441 or increasing doses (300–2,700 µM) of 2′3′-cGAMP. Graph presents the mean of three independent experiments. One-way ANOVA. (P) As in D, except that DNA-PKcs IP was incubated with or without 2′3′-cGAMP in presence or absence of NU7441 (used as a competitor) prior to measurement of bound 2′3′-cGAMP. Graph represents mean (±SEM) 2′3′-cGAMP levels; n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. (Q) FLAG-DNA-PKcs, FLAG-DNA-PKcs-Δkinase, and FLAG-kinase were expressed in 293T cells prior to TSA analysis in the presence or absence of 2′3′-cGAMP. WB was conducted with the indicated antibodies. Representative WB of three independent experiments. (R) FLAG-DNA-PKcs, FLAG-DNA-PKcs-Δkinase, and FLAG-kinase were FLAG purified as in A prior to incubation with biotin or biotinylated 2′3′-cGAMP and binding analysis by WB as in G using FLAG antibody. Representative WB of three independent experiments. ***: P < 0.001; **: P < 0.01; *: P < 0.05; ns, not significant. Also see . Source data are available for this figure: .

Article Snippet: Vero (African green monkey), HEK293T ( Homo sapiens ), and THP-1 ( H. sapiens ) cells were obtained from the American Type Culture Collection (ATCC).

Techniques: Immunoprecipitation, Incubation, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Recombinant, Purification, Transfection, Immunofluorescence, Staining, Software, In Vitro, Binding Assay

Journal: The Journal of Experimental Medicine

Article Title: DNA-PK interacts with cyclic dinucleotides and inhibits type I interferon responses

doi: 10.1084/jem.20251796

Figure Lengend Snippet: 2′3′-cGAMP interacts with the catalytic pocket of DNA-PKcs. (A) Experimental scheme for B. Whole-cell extracts (WCEs) from T98G cells were used as input for IPs using mock IgG and DNA-PKcs–specific antibodies prior to incubation with 2′3′-cGAMP and detection of bound 2′3′-cGAMP. (B) WB analysis of DNA-PKcs IP performed as in A was conducted using the indicated antibodies. Representative WB of three independent experiments. (C) Graph represents mean (±SEM; n = 3 independent experiment) 2′3′-cGAMP levels as measured in mock and DNA-PK–specific IP performed as in A. Statistical significance was calculated by two-tailed Student t test. (D) Silver staining was conducted on recombinant DNA-PKcs used in for immunoprecipitation experiments. Representative gel of three independent experiments. (E) WB analysis of TSA, conducted on WCE from THP-1 cells incubated with or without 2′3′-cGAMP or in presence or absence of NU7441. Immunoblot was performed using DNA-PKcs–, STING-, and HSP90-specific antibodies. Representative WB of three independent experiments. (F) Heatmap representation of the relative band intensities quantified from three independent experiments performed as in E. (G) Molecular modelling and docking study of 2′3′-cGAMP into DNA-PKcs. Human DNA-PKcs in ribbon representation with 2′3′-cGAMP docked in its catalytic site. (H) The docking conformation of 2′3′-cGAMP (in red spacefill representation) into the catalytic site of DNA-PKcs in the proximity of the catalytic residues (in ball and stick representation). (I) The docked conformation adopted by 2′3′-cGAMP onto the catalytic site of DNA-PKcs upon the MDSs. (J) The 2D molecular interactions diagram of 2′3′-cGAMP with the catalytic residues of DNA-PKcs. (K) Molecular modelling of ATM and ATR. DNA-PKcs superposed to the models of ATM and ATR (in red, blue, and yellow ribbon representations, respectively). (L) Close-up of the superposed active sites of ATM, ATR, and DNA-PKcs. Each of the three kinases has significant conformational differences and docking of 2′3′-cGAMP to all of them failed to return a thermodynamically viable pose (complex conformation). (M) Experimental scheme for N. Recombinant DNA-PKcs was immunoprecipitated using a DNA-PKcs–specific antibody, prior to incubation or not with increasing doses of NU7441 (0, 0.2, 2, and 20 µM) followed by 2′3′-cGAMP incubation, release of bound 2′3′-cGAMP, and detection by ELISA (N) Graph represents mean (±SEM) 2′3′-cGAMP levels as measured in DNA-PK–specific IP performed as in M. n = 3 independent experiments. Statistical significance was calculated by two-tailed Student t test. ***: P < 0.001; **: P < 0.01; *: P < 0.05; ns, not significant. Related to . Source data are available for this figure: . IP, immunoprecipitation.

Article Snippet: Vero (African green monkey), HEK293T ( Homo sapiens ), and THP-1 ( H. sapiens ) cells were obtained from the American Type Culture Collection (ATCC).

Techniques: Incubation, Two Tailed Test, Silver Staining, Recombinant, Immunoprecipitation, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Experimental Medicine

Article Title: DNA-PK interacts with cyclic dinucleotides and inhibits type I interferon responses

doi: 10.1084/jem.20251796

Figure Lengend Snippet: DNA-PKcs dampens 2′3′-cGAMP signaling. (A) T98G cells were treated or not with 2 µM NU7441 DNA-PKcs inhibitor for 1 h prior to transfection or not of 10 µg/ml 2′3′-cGAMP for 6 h and analysis of WCE by WB using indicated antibodies. Representative WB of three independent experiments. (B) As in A, except that gene expression analysis was conducted. Graphs represent mean (±SEM); n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. (C) As in A, except that IFNβ levels were measured by ELISA 6 h after treatment. Graphs represent mean (±SEM); n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. (D) T98G cells were transfected for DNA-PKcs– or KU70-targeting siRNA or a control nontargeting siRNA prior to transfection or not for 2′3′-cGAMP for 6 h and gene expression analyses. Graphs represent mean (±SEM); n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. (E) Control and DNA-PKcs knockout T98G cells were transfected with 2′3′-cGAMP for 6 h prior to gene expression analysis. Graphs present mean (±SEM); n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. (F) Control, cGAS −/− , and STING −/− THP-1 cells were treated or not with 2 µM of NU7441 for 1 h prior to transfection with 10 µg/ml 2′3′-cGAMP for 6 h and gene expression analysis. Graphs present mean (±SEM); n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. (G) As in F, except that CXCL10 levels were measured by ELISA 6 h after treatment. Graphs represent mean (±SEM); n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. (H) Control and DNA-PKcs knockout THP-1 cells were transfected with 2′3′-cGAMP for 6 h prior to gene expression analysis. Graphs present mean (±SEM); n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. (I) T98G expressing cGAS (T98G cGAS ) were transfected with dsDNA in presence or absence of NU7441. Gene expression analysis was conducted at 3, 6, 16, 24, and 48 h after transfection. Graphs present mean (±SEM) of three independent experiments. Statistical significance was calculated by two-tailed Student's t test. (J) T98G cGAS was transfected or not with dsDNA in presence or absence of NU7441 for 24 h prior to analysis of cytokines in media using cytokine arrays. Heatmap (representative of n = 2 independent experiments) represents fold change in spot intensity in NU7441-treated samples versus vehicle. ***: P < 0.001; **: P < 0.01; *: P < 0.05; ns, not significant. Also see . Source data are available for this figure: . WCE, whole-cell extracts.

Article Snippet: Vero (African green monkey), HEK293T ( Homo sapiens ), and THP-1 ( H. sapiens ) cells were obtained from the American Type Culture Collection (ATCC).

Techniques: Transfection, Gene Expression, Two Tailed Test, Enzyme-linked Immunosorbent Assay, Control, Knock-Out, Expressing

Journal: The Journal of Experimental Medicine

Article Title: DNA-PK interacts with cyclic dinucleotides and inhibits type I interferon responses

doi: 10.1084/jem.20251796

Figure Lengend Snippet: DNA-PKcs dampens 2′3′-cGAMP-mediated STING signaling. (A) Graph represents mean (±SEM, n = 3 independent experiment) CXCL10 and CCL5 levels as measured in supernatant of T98G cells treated or not with 2 µM NU7441 for 1 h prior to transfection or not of 10 µg/ml 2′3′-cGAMP for 6 h. Statistical significance was calculated by two-tailed Student's t test. (B) T98G cells were pretreated with the NU7441 (2 µM), NU7026 (10 µM), and AZD7648 (5 µM) DNA-PKcs inhibitors for 1 h prior to transfection of 2′3′-cGAMP (10 µg/ml) for 6 h and gene expression analysis. Graphs present the mean (±SEM, n = 3 independent experiments). Statistical significance was calculated by two-tailed Student's t test. (C) T98G cells were pretreated with the NU7441 (2 µM) for 1 h prior to transfection with dsDNA (2 µg) for 6 h and gene expression analysis. Graphs present the mean (±SEM, n = 3 independent experiments). Statistical significance was calculated by two-tailed Student's t test. (D) T98G cells were transfected for DNA-PKcs– or KU70-targeting siRNAs or a control nontargeting siRNA for 48 h prior to analysis of knockdown efficiency by WB using the indicated antibodies. Representative WB; n = 3 independent experiments. (E) T98G cells were engineered to express control nontargeting or PRKDC-targeting gRNA. Representative WB; n = 3 independent experiments. (F) THP-1 CTRL, THP1 cGAS−/− , and THP1 STING−/− were pretreated or not with the NU7441 (2 µM) inhibitor for 1 h prior to transfection or not of 2′3′-cGAMP (10 µg/ml) for 6 h. WB analysis was performed using the indicated antibodies. Representative WB of three to five independent experiments. (G) Densitometric quantification of band intensities of the p-IRF3/IRF3 ratio from the WB in F. Results shown as % of induction of 2′3′-cGAMP response ( n = 3–5 independent experiments). (H) Densitometric quantification of band intensities of the pSTING/STING ratio from the WB in F. Results shown as % of induction of 2′3′-cGAMP response ( n = 3–5 independent experiments). (I) Graph represents mean (±SEM, n = 3 independent experiment) IFNβ levels as measured in supernatant of THP1 CTRL , THP1 cGAS−/− , and THP1 STING−/− pretreated or not with the NU7441 (2 µM) inhibitor for 1 h prior to transfection or not of 2′3′-cGAMP (10 µg/ml) for 6 h. Statistical significance was calculated by two-tailed Student's t test. (J) T98G cells were engineered to express control nontargeting or PRKDC-targeting gRNA. Representative WB of three independent experiments. (K) T98G cells engineered to express control nontargeting or STING-targeting gRNAs were treated or not with 2 µM of NU7441 prior to transfection or not of 10 µg/ml 2′3′-cGAMP and gene expression analysis. Graphs present the mean (±SEM, n = 5 independent experiments). Statistical significance was calculated by two-tailed Student's t test. (L) As in K, except that WB analysis was performed using the indicated antibodies. Representative WB of three independent experiments. (M) As in L, except that T98G cells expressing an IFNAR-targeting gRNA were used. Representative WB of three independent experiments. (N) THP-1 cells were pretreated or not with the NU7441 (2 µM) inhibitor for 1 h prior to transfection with dsDNA (2 µg) for up to 24 h. Gene expression analysis was conducted at 3, 6, 16, and 24 h. Graphs present the mean (±SEM, n = 3 independent experiments). Statistical significance was calculated by two-tailed Student's t test. (O) Cell culture supernatants were collected at 24 h in experiment performed as in N, and cytokine/chemokine levels were analyzed using a proteome profiler array. Heatmap representation of relative spot intensities is shown (mean of three independent experiments). (P) STING-deficient THP-1 cells engineered to express human STING haplotypes (STING-H232, STING-AQ, and STING-HAQ) were pretreated or not with the NU7441 (2 µM) for 1 h prior to transfection of 2′3′-cGAMP (10 µg/ml) for 6 h and gene expression analysis. Graphs present the mean (±SEM, n = 3 independent experiments). Statistical significance was calculated by two-tailed Student's t test. (Q) As in P, except that IFNβ and CXCL10 levels were quantified by ELISA in supernatants. Graphs present the mean (±SEM, n = 3 independent experiments). Statistical significance was calculated by two-tailed Student's t test. ****: P < 0.0001; ***: P < 0.001; **: P < 0.01; *: P < 0.05; ns, not significant. Related to . Source data are available for this figure: .

Article Snippet: Vero (African green monkey), HEK293T ( Homo sapiens ), and THP-1 ( H. sapiens ) cells were obtained from the American Type Culture Collection (ATCC).

Techniques: Transfection, Two Tailed Test, Gene Expression, Control, Knockdown, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Experimental Medicine

Article Title: DNA-PK interacts with cyclic dinucleotides and inhibits type I interferon responses

doi: 10.1084/jem.20251796

Figure Lengend Snippet: DNA-PKcs inhibits 3′3′-cGAMP- and agonist-associated STING activation. (A) ATP hydrolysis by DNA-PK was measured in vitro in the presence of increasing doses (0.8–2,500 µM) of 3′3′-cGAMP or c-di-AMP. Graphs present the mean of three independent experiments. Statistical significance was calculated by one-way ANOVA. (B) Recombinant DNA-PKcs was immunoprecipitated using either mock IgG or a DNA-PKcs–specific antibody prior to incubation with 3′3′-cGAMP or c-di-AMP and ELISA-based measurement of bound CDNs. Graph presents mean (±SEM) 3′3′-cGAMP and c-diAMP levels as measured in mock and DNA-PKcs–specific IP in three independent experiments. Statistical significance was calculated by two-tailed Student's t test. (C) THP-1 cells were treated or not with 2 μM NU7441 in combination or not with 10 µg/ml fluorinated 3′3′-cGAMP for 6 h prior to WB analysis using the indicated antibodies. Representative WB of three independent experiments. (D) As in C, except that gene expression analyses were conducted. Graphs present the mean (±SEM) of three independent experiments. Statistical significance was calculated by two-tailed Student's t test. (E) As in C, except that IFNβ, CXCL10, and CCL5 levels were measured by ELISA. Graphs present the mean (±SEM) of three independent experiments. Statistical significance was calculated by two-tailed Student's t test. (F) Control and DNA-PKcs knockout THP-1 cells were treated with 3′3′-cGAMP for 6 h prior to gene expression analysis. Graphs present mean (±SEM); n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. (G) Human primary monocytes were isolated from buffy coats prior to treatment or not with 2 µM NU7441 for 1 h, followed by administration of 10 µg/ml fluorinated 3′3′-cGAMP for 6 h and gene expression analysis. Graphs present the mean (±SEM) of three independent experiments. Statistical significance was calculated by two-tailed Student's t test. (H) ATP hydrolysis by DNA-PKcs was measured in vitro in presence of increasing doses (0.8–2,500 µM) of E7766 or ADU-S100. Statistical significance was calculated by one-way ANOVA. (I) T98G cells were treated or not with 2 µM of NU7441 prior to addition or not of 1 µM of E7766 STING agonist for 3 h and gene expression analysis. Graphs present the mean (±SEM); n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. (J) T98G cells were treated or not with 2 µM of NU7441 prior to addition or not of 50 µM of ADU-S100 STING agonist for 3 h and gene expression analysis. Graphs present the mean (±SEM), n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. (K) T98G cells were treated or not with 2 µM of NU7441 prior to addition or not of 10 µM of diABZI for 3 h and gene expression analysis. Graphs present the mean (±SEM); n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. ****: P < 0.0001; ***: P < 0.001; **: P < 0.01; *: P < 0.05; ns, not significant. Also see . IP, immunoprecipitation.

Article Snippet: Vero (African green monkey), HEK293T ( Homo sapiens ), and THP-1 ( H. sapiens ) cells were obtained from the American Type Culture Collection (ATCC).

Techniques: Activation Assay, In Vitro, Recombinant, Immunoprecipitation, Incubation, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Gene Expression, Control, Knock-Out, Isolation

Journal: The Journal of Experimental Medicine

Article Title: DNA-PK interacts with cyclic dinucleotides and inhibits type I interferon responses

doi: 10.1084/jem.20251796

Figure Lengend Snippet: DNA-PKcs selectively counteracts CDNs. (A) ATP hydrolysis by DNA-PKcs was measured in vitro in presence of increasing doses (0.8–2,500 µM) of c-di-GMP. Graph presents the mean (±SEM) performed in biological triplicates. Statistical significance was calculated by one-way ANOVA. ns, not significant. (B) T98G cells were treated or not with 2 μM NU7441 in combination or not with 10 µg/ml fluorinated 3′3′-cGAMP for 6 h prior to WB analysis using the indicated antibodies. Representative WB of three independent experiments. (C) As in B, except that gene expression analyses were conducted. Graphs present the mean (±SEM) performed in biological triplicates. Statistical significance was calculated by two-tailed Student's t test. ***: P < 0.001; **: P < 0.01; *: P < 0.05. (D) Experimental scheme for human primary monocyte isolation and treatment . Human primary monocytes were isolated from buffy coats prior to treatment or not with 2 µM NU7441 for 1 h, followed by administration of 10 µg/ml fluorinated 3′3′-cGAMP for 6 h and gene expression analysis. (E) Flow cytometry analysis of macrophages prepared as in . (F) Gene expression analyses were performed on human primary cells treated as described in . Graphs present the mean (±SEM) expression of IFIT1 in three independent experiments. Statistical significance was calculated by two-tailed Student's t test. (G) T98G cells were treated or not with 2 µM of NU7441 prior to addition or not of 1 µM of E7766 STING agonist for 3 h and analysis of gene expression. WB analyses were conducted using indicated antibodies and are representative of three independent experiments. (H) T98G cells were treated or not with 2 µM of NU7441 prior to addition or not of 50 µM of ADU-S100 STING agonist for 3 h and analysis of gene expression. WB analyses were conducted using indicated antibodies and are representative of three independent experiments. (I) T98G cells were treated or not with 2 µM of NU7441 prior to addition or not of 10 µM of diABZI for 3 h and analysis of gene expression. WB analyses were conducted using indicated antibodies and are representative of three independent experiments. (J) Control and DNA-PKcs knockout THP-1 cells were treated with 1 µM E7766 for 6 h prior to gene expression analysis. Graphs present mean (±SEM), n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. (K) Control and DNA-PKcs knockout THP-1 cells were treated with 10 µM diABZI for 6 h prior to gene expression analysis. Graphs present mean (±SEM); n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. ****: P < 0.0001; ***: P < 0.001; **: P < 0.01; *: P < 0.05. Related to . Source data are available for this figure: .

Article Snippet: Vero (African green monkey), HEK293T ( Homo sapiens ), and THP-1 ( H. sapiens ) cells were obtained from the American Type Culture Collection (ATCC).

Techniques: In Vitro, Gene Expression, Two Tailed Test, Isolation, Flow Cytometry, Expressing, Control, Knock-Out

Journal: The Journal of Biological Chemistry

Article Title: Improvement in binding and function of a monoclonal antibody against Shigella flexneri 3a O-antigen via phage display and whole-cell in-solution panning

doi: 10.1016/j.jbc.2026.111405

Figure Lengend Snippet: Functional analysis of hFlex3a2_v2 variants against S. flexneri 3a. A , L-ABA assay comparing the ability of single hFlex3a2_v2 variants or ( B ) combinatorial hFlex3a2_v2 variants and a non-binding control to coordinate complement-mediated bacteriolysis of S. flexneri 3a. L-ABA is the average of three biological replicates each performed in duplicate. L-ABAs in panels ( A ) and ( B ) were performed in parallel but graphed separately for clarity. hFlex3a2_v2 and control mAb data is the same in these two panels. p values in were calculated on logIC 50 values by repeated measure Anova with Dunnett’s correction for multiple comparisons. C , OPA assay measures the percentage of THP-1 cells that have phagocytosed S. flexneri 3a that was opsonized with 30 nM of the indicated mAb. This experiment is the result of five biological replicates each performed in duplicate, and p values were calculated by repeated measure ANOVA with Dunnett’s correction for multiple comparisons. Error bars represent standard deviation. D , Invasion assay shows the percent invasion of S. flexneri 3a incubated with 1 μM of the indicated mAb normalized to the invasion rate of S. flexneri 3a in PBS. This experiment is the result of four biological replicates each performed in duplicate, and p -values were calculated by repeated measure ANOVA with Dunnett’s correction for multiple comparisons. Error bars represent standard deviation. For all data, p values can be found in and those < 0.5 are denoted by a ∗. ( E ) A cartoon depicting the top-ranked Alphafold model of hFlex3a2_v2 (pLDDT = 96. 2, pTM = 0.929, and ipTM = 0.909). The VL is shown in light gray, and the VH is shown in dark gray. CDRL loops are colored orange, and residues where variants were selectively enriched against S. flexneri 3a following panning against S. flexneri 3a and S. sonnei and were experimentally characterized are colored green , while H34 (not selectively enriched) is shown in pink .

Article Snippet: THP-1 cell lines were obtained from ATCC.

Techniques: Functional Assay, Binding Assay, Control, OPA Assay, Standard Deviation, Invasion Assay, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Improvement in binding and function of a monoclonal antibody against Shigella flexneri 3a O-antigen via phage display and whole-cell in-solution panning

doi: 10.1016/j.jbc.2026.111405

Figure Lengend Snippet: Functional analysis of hFlex3a2_v2 variants against S. flexneri 3b. Binding curves from a titration ELISA against S. flexneri 3b OMVs (2.5 μg/ml) show relative binding potencies of hFlex3a2_v2 ( A ) single amino acid variants or ( B ) hFlex3a2_v2 combinatorial amino acid variants. ELISA binding curves are the average of three independent replicates each performed in duplicate. ELISAs in panels ( A ) and ( B ) were performed in parallel but graphed separately for clarity. hFlex3a2_v2 and control mAb data is the same in these two panels. ( C ) L-ABA assay comparing the ability of single hFlex3a2_v2 variants or ( D ) combinatorial hFlex3a2_v2 variants and a non-binding control to coordinate complement-mediated bacteriolysis of S. flexneri 3b. L-ABA is the average of three biological replicates each performed in duplicate. L-ABAs in panels ( C ) and ( D ) were performed in parallel but graphed separately for clarity. hFlex3a2_v2 and control mAb data is the same in these two panels. p -values were calculated on logIC 50 values by repeated measure Anova with Dunnett’s correction for multiple comparisons ( E ) OPA assay measures the percent of THP-1 cells that have phagocytosed S. flexneri 3b that was opsonized with 30 nM of the indicated mAb. This experiment is the result of five biological replicates each performed in duplicate, and p -values were calculated by repeated measure ANOVA with Dunnett’s correction for multiple comparisons. Error bars represent standard deviation. F , invasion assay shows the percent invasion of S. flexneri 3b incubated with 1 μM of the indicated mAb normalized to the invasion rate of S. flexneri 3b in PBS. This experiment is the result of four biological replicates each performed in duplicate. Error bars represent standard deviation. For all plots, p values can be found in and those < 0.5 are denoted by a ∗.

Article Snippet: THP-1 cell lines were obtained from ATCC.

Techniques: Functional Assay, Binding Assay, Titration, Enzyme-linked Immunosorbent Assay, Control, OPA Assay, Standard Deviation, Invasion Assay, Incubation

Journal: Translational Oncology

Article Title: Unmasking FCGR2B as a high-grade serous ovarian cancer specific marker of immune suppression and tumor progression through multi-omics mining

doi: 10.1016/j.tranon.2026.102748

Figure Lengend Snippet: FCGR2B expression defines an immunosuppressive TME in HGSOC. (A) ssGSEA-based immune infiltration analysis across 28 immune cell types. Δ = High - Low. (B) CIBERSORT-based macrophage-focused immune deconvolution reveals distinct immune landscapes between FCGR2B -High and FCGR2B -Low tumors. (C) Fluorescence images (scale bar, 50 μm) of FCGR2B (red), CD14 (green), CD68 (yellow), and the corresponding merged image. (D-E) Quantitative analysis of FCGR2B co-localization with CD14 (D) and CD68 (E). Left: box plots comparing co-localization levels between FCGR2B-High and FCGR2B-Low tumors (defined by the median FCGR2B H-score; Wilcoxon rank-sum test). Right: scatter plots showing correlations between FCGR2B and marker H-scores across all HGSOC samples (Spearman’s rho). (F) FCGR2B protein expression in HGSOC cell lines (SKOV3 and OVCAR3) and the cell line THP-1, as detected by WB assays. (G) FCGR2B protein expression in THP-1 cells transfected with Si-FCGR2B or control siRNA, as detected by WB assays. (H) Effect of FCGR2B knockdown on macrophage M2 polarization (CD206⁺/CD68⁺) under SKOV3 (HGSOC) co-culture conditions, assessed by flow cytometry. (I) Effect of FCGR2B knockdown on macrophage phagocytosis (CD68⁺/CFSE⁺) of SKOV3 cells under co-culture conditions, analyzed by flow cytometry. Statistical significance was determined using a two-tailed unpaired Student’s t -test; exact P values are indicated within the figure ( P = 0.0001, t = 14.35 for H; P = 0.0013, t = 7.976 for I; n =3, df = 4).

Article Snippet: The HGSOC cell lines SK-OV-3 (ATCC® HTB-77TM) and OVCAR-3 (ATCC® HTB-161TM), as well as the human monocytic cell line THP-1 (ATCC® TIB-202TM), were obtained from the American Type Culture Collection and cultured under standard conditions at 37 °C with 5% CO2.

Techniques: Expressing, Fluorescence, Marker, Transfection, Control, Knockdown, Co-Culture Assay, Flow Cytometry, Two Tailed Test