Journal: Journal of Advanced Research

Article Title: Enhancement of NK cell activity via DNA-sensing inhibition by Poxin transgene

doi: 10.1016/j.jare.2025.05.058

Figure Lengend Snippet: Poxin transgene inhibits DNA sensing-induced innate immune response. A. Schematic of cloning process. B-G. Cells were transfected with plasmid DNA (pDNA) using TransIT-2020. B. 2.5 μg and 7.5 μg of pDNA encoding Poxin or mutant Poxin (mtPoxin) were transfected into 293T cells, respectively, and FLAG-tagged proteins were detected. C. Activation of STING, TBK1, and IRF3 was analyzed by western blotting. Quantitative analysis of p-IRF3 expression level is shown. D. H151 was pre-treated 1 h before pDNA transfection, and protein expression levels were assessed after 24 h. E. Cells were transfected with pEGFP-N1, and protein expression levels were detected. F. mRNA levels of IFNB1 and ISG15 were analyzed by RT-qPCR (n = 3). G. IFN-β secretion levels were quantitatively analyzed using ELISA. (Representative western blots are shown.).

Article Snippet: The Poxin coding sequence (CDS) fragment was synthesized by Bioneer, and the H17A mutant Poxin (mtPoxin) fragment was generated via overlapping PCR.

Techniques: Cloning, Transfection, Plasmid Preparation, Mutagenesis, Activation Assay, Western Blot, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Journal of Advanced Research

Article Title: Enhancement of NK cell activity via DNA-sensing inhibition by Poxin transgene

doi: 10.1016/j.jare.2025.05.058

Figure Lengend Snippet: Poxin protects NK-92 cells from pDNA transfection-induced innate immunity. A. Cells were transfected by electroporation with 10 μg pDNA, and mRNA dynamics were analyzed by RT-qPCR (n = 3). B. IFN-β secretion levels were quantitatively analyzed using ELISA. C. Expression levels of innate immune signaling proteins were detected by western blotting. (Representative data shown). D. Quantitative analysis of p-STING and p-TBK1 expression levels.

Article Snippet: The Poxin coding sequence (CDS) fragment was synthesized by Bioneer, and the H17A mutant Poxin (mtPoxin) fragment was generated via overlapping PCR.

Techniques: Transfection, Electroporation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot

Journal: Journal of Advanced Research

Article Title: Enhancement of NK cell activity via DNA-sensing inhibition by Poxin transgene

doi: 10.1016/j.jare.2025.05.058

Figure Lengend Snippet: Poxin prolongs NK-92 cell survival and activity by reducing irradiation-induced innate immune activation. A. Cell survival rate was measured by FACS at specified times after irradiation. B. Cell proliferation rate was quantified by counting live cells. C. Cytotoxicity of effector cells against K562 cells was measured 1- and 2-days post-irradiation over a 4-hour period. D. Protein expression levels were detected by western blotting. (Representative data shown). E. Quantitative analysis of p-IRF3 expression level.

Article Snippet: The Poxin coding sequence (CDS) fragment was synthesized by Bioneer, and the H17A mutant Poxin (mtPoxin) fragment was generated via overlapping PCR.

Techniques: Activity Assay, Irradiation, Activation Assay, Expressing, Western Blot

Journal: Journal of Advanced Research

Article Title: Enhancement of NK cell activity via DNA-sensing inhibition by Poxin transgene

doi: 10.1016/j.jare.2025.05.058

Figure Lengend Snippet: Poxin transgene enhances the cytotoxic activity of NK-92 cells. A. Cytotoxicity of NK-92 cells with and without Poxin expression against K562 cells was measured over 4 h. B-C. Cells were co-cultured with target K562 cells for indicated times. B. Apoptosis levels in effector cells were assessed by FACS. C. Protein expression levels were detected by western blotting. (Representative data shown). D-G. Cells were harvested after 24 h in culture, and extracted total RNA was analyzed by RNA sequencing. (C = mock cells; P = Pox-NK-92 cells; n = 3). D. Heatmap. E. GO analysis. F. Differentially expressed genes in NK-92 and Pox-NK-92 groups were categorized by function. G. Top 10 upstream transcription factors and target genes were analyzed by network analysis. H. NK cell activation pathway was analyzed by western blotting. (Representative data shown). I. Quantitative analysis of Perforin expression level. J. mRNA levels of cytotoxic factors were analyzed by RT-qPCR (n = 3).

Article Snippet: The Poxin coding sequence (CDS) fragment was synthesized by Bioneer, and the H17A mutant Poxin (mtPoxin) fragment was generated via overlapping PCR.

Techniques: Activity Assay, Expressing, Cell Culture, Western Blot, RNA Sequencing, Activation Assay, Quantitative RT-PCR

Journal: Journal of Advanced Research

Article Title: Enhancement of NK cell activity via DNA-sensing inhibition by Poxin transgene

doi: 10.1016/j.jare.2025.05.058

Figure Lengend Snippet: Poxin enhances the anti-tumor capacity of NK-92 cells. A. Experimental scheme for mouse study (n = 4). B. Ventral bioluminescence (BLI) images following injection of NK92 or Pox-NK-92 cells. C. Quantification and statistical analysis (n = 4). D. Survival curves post-injection of K562-Luc cells. Data are presented as mean ± SEM. Mouse experiments were conducted independently twice, yielding consistent results.

Article Snippet: The Poxin coding sequence (CDS) fragment was synthesized by Bioneer, and the H17A mutant Poxin (mtPoxin) fragment was generated via overlapping PCR.

Techniques: Injection

Journal: Journal of Advanced Research

Article Title: Enhancement of NK cell activity via DNA-sensing inhibition by Poxin transgene

doi: 10.1016/j.jare.2025.05.058

Figure Lengend Snippet: Poxin improves the anti-tumor efficacy of PD-L1-CAR-NK cells against H460 lung cancer. A. PD-L1-CAR expression levels in NK-92 cells. Abbreviations: TM = Transmembrane; Co-stim=Co-stimulatory; SD=Signaling domain. B. Cytotoxicity of CAR-NK-92 cells with or without Poxin expression against K562 and H460 cells, measured over 2 h. C. Experimental scheme for the H460 xenograft mouse model (n = 8). D. Tumor sizes were measured using digital calipers at the indicated time points. E. Representative tumor images from both groups at 28 days post-H460 inoculation. F. Tumor volumes were calculated. Error bars in panels B and D represent mean ± SEM. Mouse experiments were independently repeated twice with similar outcomes. Statistical significance was assessed using Two-way ANOVA (D) or Student's t -test (F).

Article Snippet: The Poxin coding sequence (CDS) fragment was synthesized by Bioneer, and the H17A mutant Poxin (mtPoxin) fragment was generated via overlapping PCR.

Techniques: Expressing

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: IRF1 suppresses gastric tumorigenesis via dual PI3K/AKT-ERK pathway modulation and functional antagonism of oncogenic MX2

doi: 10.1007/s13402-025-01134-w

Figure Lengend Snippet: Expression of IRF1 and Type I interferon pathway-related proteins in gastric cancer cells. A : Simulation of type I interferon response in gastric cancer cells by incubating with interferon, showing increased expression of interferon pathway protein IRF1. B : Protein expression in gastric cancer cells after transfection with pcDNA3.1(+) -IRF1-HA.1 C : Protein expression in gastric cancer cells after transfection with siRNA-IRF1. D : Changes in the expression of interferon pathway proteins after overexpression or inhibition of IRF1 in gastric cancer cells

Article Snippet: The IRF1 gene coding sequence (CDS) was retrieved from the National Center for Biotechnology Information (NCBI), selecting the longest transcript.

Techniques: Expressing, Transfection, Over Expression, Inhibition

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: IRF1 suppresses gastric tumorigenesis via dual PI3K/AKT-ERK pathway modulation and functional antagonism of oncogenic MX2

doi: 10.1007/s13402-025-01134-w

Figure Lengend Snippet: Expression characteristics and clinical relevance of IRF1 in gastric cancer tissues. A : IRF1 expression is significantly higher in gastric cancer tissues compared to adjacent normal tissues ( P < 0.001). B : Paired analysis shows that IRF1 expression is significantly upregulated in gastric cancer tissues compared to matched adjacent tissues ( P = 0.002). C : Survival analysis indicates that high IRF1 expression is associated with a relatively better prognosis in gastric cancer patients (Log-rank P = 0.030). D : Representative immunohistochemistry (IHC) images showing high and low IRF1 expression in tumor tissues. E : Differential expression analysis confirms that IRF1 is significantly upregulated in tumor tissues ( P = 0.021). F : Paired analysis of tumor and adjacent normal tissues demonstrates significantly higher IRF1 expression in tumors ( P < 0.001). G : Survival analysis further supports that high IRF1 expression correlates with a better prognosis in gastric cancer patients (Log-rank P = 0.010). * P < 0.05, ** P < 0.01, *** P < 0.001, *** P < 0.0001

Article Snippet: The IRF1 gene coding sequence (CDS) was retrieved from the National Center for Biotechnology Information (NCBI), selecting the longest transcript.

Techniques: Expressing, Immunohistochemistry, Quantitative Proteomics

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: IRF1 suppresses gastric tumorigenesis via dual PI3K/AKT-ERK pathway modulation and functional antagonism of oncogenic MX2

doi: 10.1007/s13402-025-01134-w

Figure Lengend Snippet: Effects of IRF1 expression on gastric cancer cell function. A - B : CCK8 cytotoxicity assay showing inhibition of AGS and BGC-823 cell proliferation after transfection with IRF1-HA overexpression plasmid. C - D : Transwell migration and invasion assay showing suppression of AGS and BGC-823 cell migration and invasion 48 h after transfection with IRF1-HA overexpression plasmid. E - F : CCK8 cytotoxicity assay showing promotion of AGS and BGC-823 cell proliferation after transfection with siRNA-IRF1. G - H : Transwell migration and invasion assay showing enhanced migration and invasion of AGS and BGC-823 cells 48 h after transfection with siRNA-IRF1. I : Flow cytometry apoptosis assay showing increased apoptosis in gastric cancer cells 48 h after Dox-induced IRF1 overexpression in stable cell lines. J : Changes in apoptosis-related protein expression 48 h after Dox-induced IRF1 overexpression in gastric cancer cell stable lines. K : Immunofluorescence localization experiment confirming nuclear localization of IRF1 in gastric cancer cells. Scale bar: 10 μm. * P < 0.05, ** P < 0.01, ** P < 0.001

Article Snippet: The IRF1 gene coding sequence (CDS) was retrieved from the National Center for Biotechnology Information (NCBI), selecting the longest transcript.

Techniques: Expressing, Cell Function Assay, Cytotoxicity Assay, Inhibition, Transfection, Over Expression, Plasmid Preparation, Migration, Invasion Assay, Flow Cytometry, Apoptosis Assay, Stable Transfection, Immunofluorescence

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: IRF1 suppresses gastric tumorigenesis via dual PI3K/AKT-ERK pathway modulation and functional antagonism of oncogenic MX2

doi: 10.1007/s13402-025-01134-w

Figure Lengend Snippet: Establishment of an Inducible IRF1 expression nude mouse subcutaneous xenograft and lung metastasis model. A : In vivo experiments show that the tumor volume of the IRF1 overexpression group in nude mouse subcutaneous xenografts is significantly smaller than that of the control group (Mean ± SEM). B : Gross anatomy of subcutaneous xenograft tumors in nude mice. C : HE staining of subcutaneous xenograft tumors (tumor tissues from mouse #1 and #2). D : Immunohistochemistry (IHC) analysis of IRF1 expression in subcutaneous xenograft tumors, showing significant IRF1 overexpression in the Dox (+) group. E : Bioluminescence imaging of nude mice injected via the tail vein with BGC-IRF1-LUC cells at 0, 2, 3, and 4 weeks post-injection. F : Quantification of lung fluorescence intensity in both groups, demonstrating significantly lower fluorescence intensity in the IRF1 overexpression group compared to the control group. G : Gross anatomy of lung tissues in nude mice, showing more metastatic nodules in the control group than in the IRF1 overexpression group. H : HE staining and IRF1-specific IHC staining of lung tissues in nude mice, confirming IRF1 overexpression in the Dox-induced group. Scale bar: 100 μm. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The IRF1 gene coding sequence (CDS) was retrieved from the National Center for Biotechnology Information (NCBI), selecting the longest transcript.

Techniques: Expressing, In Vivo, Over Expression, Control, Staining, Immunohistochemistry, Imaging, Injection, Fluorescence

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: IRF1 suppresses gastric tumorigenesis via dual PI3K/AKT-ERK pathway modulation and functional antagonism of oncogenic MX2

doi: 10.1007/s13402-025-01134-w

Figure Lengend Snippet: Exploration of IRF1-related mechanisms. A : Schematic diagram of the IRF1 protein structure; DBD: DNA binding domain; IAD: IRF-association domain. B : Validation of successful expression of the pcDNA3.1(+)-IRF1Δ1 recombinant plasmid. C : No significant changes in apoptosis-related protein expression after IRF1Δ1 overexpression. D : Overexpression of IRF1 significantly affects PI3K pathway-related proteins, whereas IRF1Δ1 overexpression shows no apparent changes. E : Bidirectional validation of the interaction between IRF1 and MX2 using HA and Flag magnetic beads. F : Immunofluorescence co-localization analysis confirming the nuclear co-expression of IRF1 and MX2

Article Snippet: The IRF1 gene coding sequence (CDS) was retrieved from the National Center for Biotechnology Information (NCBI), selecting the longest transcript.

Techniques: Binding Assay, Biomarker Discovery, Expressing, Recombinant, Plasmid Preparation, Over Expression, Magnetic Beads, Immunofluorescence

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: IRF1 suppresses gastric tumorigenesis via dual PI3K/AKT-ERK pathway modulation and functional antagonism of oncogenic MX2

doi: 10.1007/s13402-025-01134-w

Figure Lengend Snippet: MX2-related functions in gastric cancer. A : Schematic diagram of MX2 functional domain fragments. B : Co-IP assay validating the interaction between IRF1-HA and MX2-Flag, showing that IRF1 loses interaction with MX2-FlagΔ1. C : Schematic of MX2 site-directed mutations. K131A: GTP-binding defective, unable to enter the nucleus; NLS: nuclear localization signal; BSE: bundle signaling element. D : WB validation of MX2 site-directed mutant overexpression plasmids. E : MX2 overexpression promotes AGS cell migration and invasion. F : MX2 overexpression promotes BGC-823 cell migration and invasion. G : After transfecting MX2 and its mutants into BGC-823 cells and incubating for 48 h, nuclear localization site mutations in MX2 result in the loss of its pro-migratory and pro-invasive effects. H : Immunofluorescence localization of MX2 and its mutants in gastric cancer cells. After transfection with MX2-ΔN or MX2-K131A, MX2 is retained in the cytoplasm, losing its nuclear localization ability. I : WB validation of MX2’s pro-migratory function through EMT pathway analysis. Scale bar: 10 μm. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The IRF1 gene coding sequence (CDS) was retrieved from the National Center for Biotechnology Information (NCBI), selecting the longest transcript.

Techniques: Functional Assay, Co-Immunoprecipitation Assay, Binding Assay, Biomarker Discovery, Mutagenesis, Over Expression, Migration, Immunofluorescence, Transfection

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: IRF1 suppresses gastric tumorigenesis via dual PI3K/AKT-ERK pathway modulation and functional antagonism of oncogenic MX2

doi: 10.1007/s13402-025-01134-w

Figure Lengend Snippet: Functional model of the IRF1-MX2 protein complex

Article Snippet: The IRF1 gene coding sequence (CDS) was retrieved from the National Center for Biotechnology Information (NCBI), selecting the longest transcript.

Techniques: Functional Assay