Journal: Translational Oncology

Article Title: REST-driven upregulation of SFXN3 promotes AML progression via Wnt/β-catenin activation and confers decitabine resistance

doi: 10.1016/j.tranon.2026.102705

Figure Lengend Snippet: REST Regulates SFXN3 Expression by Directly Binding to Its Promoter Region. (A) Identification of transcription factors potentially regulating SFXN3 expression by integrating data from the JASPAR, GTEx, ChIP Atlas, and ENCODE databases. A Venn diagram highlights TFAP2C and REST as common candidates, suggesting them as potential upstream regulators. (B) Quantitative mRNA analysis reveals that REST is significantly upregulated in AML tissues compared to adjacent non-tumor tissues. (C) Spearman correlation analysis shows a strong positive correlation between REST and SFXN3 expression. (D) RT-PCR analysis confirms the knockdown efficiency of two shRNA constructs targeting REST (sh-REST#1 and sh-REST#2), and evaluates their impact on SFXN3 mRNA levels. (E) Western blot analysis validates the protein-level knockdown efficiency of sh-REST#1 and sh-REST#2, along with their effects on SFXN3 protein expression. (F) Quantification of REST and SFXN3 protein expression levels. (G) Prediction of REST binding sites in the SFXN3 promoter using JASPAR and ENCODE databases, with the highest affinity region localized between positions −415 and −395. (H) Binding motif analysis from the JASPAR database confirms a predicted REST binding site within the SFXN3 promoter. A mutant version of this binding site was designed for subsequent experiments. (I) ChIP-seq data demonstrates a prominent REST binding peak at the −415 to −395 region of the SFXN3 promoter. (J) Molecular docking simulation reveals that the DNA-binding domain of REST forms a stable complex with the specific binding site in the SFXN3 promoter. (K) ChIP-qPCR analysis confirms significant enrichment of REST at the SFXN3 promoter, which is markedly reduced following REST knockdown. (L) Dual-luciferase reporter assays show that REST significantly enhances the activity of the wild-type SFXN3 promoter, while mutation of the binding site substantially diminishes this effect.

Article Snippet: Subsequently, luciferase activity was measured using a Dual-Luciferase Reporter Assay Kit (#DL101, Vazyme, Nanjing, China).

Techniques: Expressing, Binding Assay, Reverse Transcription Polymerase Chain Reaction, Knockdown, shRNA, Construct, Western Blot, Mutagenesis, ChIP-sequencing, ChIP-qPCR, Luciferase, Activity Assay

Journal: PLOS Pathogens

Article Title: The CD97-PPM1G axis dampens antiviral immunity by dephosphorylating IRF7 in type I interferon pathway

doi: 10.1371/journal.ppat.1014032

Figure Lengend Snippet: (A) Co-IP analysis showing the interaction between PPM1G and adaptors (MAVS, TBK1, IRF3, and IRF7) following transfection with the indicated plasmids and using Flag magnetic beads in HEK-293T cells. (B and C) Reverse validation of the interaction between PPM1G and IRF7/IRF3 was performed using Co-IP with HA magnetic beads. (D and E) GST pull-down analysis of the direct interaction between PPM1G and IRF7/IRF3. (F) BHK-21 cells were transfected with different doses of PPM1G (0.6, 1.2, 1.8 µg per 2.5mL in a 6-well plate) after BEFV infection 24 h to analyze the phosphorylation of IRF7/IRF3 by immunoblotting. (G) HEK-293T cells were transfected with PPM1G and its enzymatically inactive mutant (D496A) to assess the phosphorylation of IRF7/IRF3 following IKKε transfection. (H-K) Dual-luciferase reporter assays were used to evaluate the impact of (H and I) IRF7-induced or (J and K) IRF3-induced promoter activity of IFN-β and ISRE in the presence of PPM1G and its D496A mutant. This was achieved through the transfection of pRL-TK, pIFNβ-Luc, pISRE-Luc, and the indicated plasmids. The results show the mean ± SEM from three independent experiments. ns, not significant, *, p < 0.05; **, p < 0.01; ***, p < 0.001. (L) The synergistic effect of CD97 and PPM1G on IRF7 phosphorylation during VSV infection was detected by immunoblot. (M) The binding of endogenous PPM1G to IRF7 in HeLa cells infected with VSV was determined by immunoprecipitation using anti-PPM1G (or an IgG isotype control). (N) The effect of CD97 overexpression on the phosphorylation and translocation of IRF7 was evaluated through nuclear and cytoplasmic separation. Data in (A-G, L-N) are representative of three independent experiments.

Article Snippet: After 24 hours, the cells were harvested, and firefly and Renilla luciferase activities were measured using a dual-luciferase reporter assay system kit (DD1205-01; Vazyme Biotech) and a SpectraMax M5 microplate reader, following the manufacturer’s instructions.

Techniques: Co-Immunoprecipitation Assay, Transfection, Magnetic Beads, Biomarker Discovery, Infection, Phospho-proteomics, Western Blot, Mutagenesis, Luciferase, Activity Assay, Binding Assay, Immunoprecipitation, Control, Over Expression, Translocation Assay

Journal: PLOS Pathogens

Article Title: The CD97-PPM1G axis dampens antiviral immunity by dephosphorylating IRF7 in type I interferon pathway

doi: 10.1371/journal.ppat.1014032

Figure Lengend Snippet: (A) RT‒qPCR analysis of PPM1G transcripts in BHK-21 cells overexpressing CD97 following BEFV infection. (B) Immunoblotting analysis of PPM1G expression in BHK-21 cells either mock-treated or infected with BEFV. (C and D) The expression of PPM1G was analyzed in CD97 knockout HeLa cells and (D) PMs, either mock-treated or infected with VSV. (E) PPM1G promoter truncation mutant (M1 to M5) model. (F) Various truncated PPM1G promoter constructs and a Renilla luciferase reporter vector were transfected into HEK-293T cells, and then analyzed for dual luciferase activity. (G) A heatmap of transcriptome analysis for samples with CD97 overexpression and BEFV infection in BHK-21 cells. (H) Model showing the interaction site between the indicated transcription factors and the PPM1G promotor region. (I and J) The indicated protein expression was analyzed in BHK-21 cells overexpressing CD97, either mock-treated or infected with BEFV or VSV. (K) PPM1G-M5 promoter constructs and IKZF1, along with a Renilla luciferase reporter vector, were transfected into HEK-293T cells, followed by analysis of dual luciferase activity. (L and M) RT‒qPCR and immunoblotting analysis of PPM1G expression were performed in HeLa cells overexpressing IKZF1 following VSV infection. (N) RT‒qPCR analysis of PPM1G mRNA transcripts was conducted in CD97-overexpressing HeLa cells with IKZF1 silencing. Data in (B-D, I, J, M) are representative of three independent experiments.

Article Snippet: After 24 hours, the cells were harvested, and firefly and Renilla luciferase activities were measured using a dual-luciferase reporter assay system kit (DD1205-01; Vazyme Biotech) and a SpectraMax M5 microplate reader, following the manufacturer’s instructions.

Techniques: Infection, Western Blot, Expressing, Knock-Out, Mutagenesis, Construct, Luciferase, Plasmid Preparation, Transfection, Activity Assay, Over Expression