multiple displacement amplification  (Qiagen)

Journal: Cell Reports Medicine

Article Title: Enalaprilat reverses neutrophil polarization imbalance via targeting taurine-STING axis for treatment of diabetic wounds

doi: 10.1016/j.xcrm.2026.102714

Figure Lengend Snippet: Phenotype repolarization of neutrophils induced by Ena affected the pro-angiogenic capacities of HUVECs and inflammatory state of macrophages (A) Flow cytometry was adopted to reveal the difference of CD206+ population ratio among the three groups. n = 3 independent experiments. (B) Effects of AGE stimulation and sequential Ena supplement on the expression of ICAM1, CXCR2, CXCR4, and Fas as evaluated by western blot. (C) Effects of AGE stimulation and sequential Ena supplement on the expression of ICAM1, CXCR2, CXCR4, and Fas as evaluated by immunofluorescence staining. Scale bar, 2 μm; n = 3 independent experiments. (D) Quantitative reverse-transcription PCR (RT-qPCR) was employed to quantify the level of il1b , ccl3 , arg1 , and ccl17 . n = 3 independent experiments. (E) Schematic diagram for the incubation of HUVECs with pretreated neutrophils. (F) Proliferative ability of HUVECs under the stimulation of neutrophils with indicated preconditioning as detected via EdU staining. Scale bar, 200 μm; n = 3 independent experiments. (G) Migration property of HUVECs as measured by transwell assay. Scale bar, 200 μm; n = 3 independent experiments. (H) Migration property of HUVECs as measured by wound scratch test. Scale bar, 500 μm. (I) Tube formation assay was performed to visualize the neovascularization function of HUVECs with different treatments. Scale bar, 200 μm; n = 3 independent experiments. (J) Phenotype characters of macrophages irritated by neutrophils as determined by immunofluorescence staining. Scale bar, 5 μm. (K) Phenotype characters of macrophages irritated by neutrophils as determined by flow cytometry. n = 3 independent experiments. Data were shown as mean ± standard deviation (SD) from biological replicates, and statistical analyses were performed using one-way ANOVA test followed by Tukey’s multiple comparisons test in (A–D and F–K). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Taq Pro U + multiple probe qPCR mix kit , Vazyme , Cat#QN213-01.

Techniques: Flow Cytometry, Expressing, Western Blot, Immunofluorescence, Staining, Reverse Transcription, Quantitative RT-PCR, Incubation, Migration, Transwell Assay, Tube Formation Assay, Standard Deviation

Journal: Cell Reports Medicine

Article Title: Enalaprilat reverses neutrophil polarization imbalance via targeting taurine-STING axis for treatment of diabetic wounds

doi: 10.1016/j.xcrm.2026.102714

Figure Lengend Snippet: Regulatory roles of Ena in ZNF460-GGT1 axis-mediated taurine metabolism (A) KEGG pathway analysis of taurine metabolism. (B) Heatmap of top 40 significant DEGs. (C) Predicted binding sites of ZNF460 in the promoter of GGT1 . (D) Combination of ZNF460 with the promoter sequences of GGT1 as validated by ChIP assay. n = 3 independent experiments. (E) Dual luciferase reporter gene assay was conducted to evaluate the inhibitory effects of Ena on the conjunction of ZNF460 with GGT1 promoter. n = 3 independent experiments. (F) Molecular docking analysis between Ena and ZNF460. (G) Results of cellular thermal shift assay as visualized by western blot. n = 3 independent experiments. (H) Results of drug affinity responsive target stability test as visualized by western blot. n = 3 independent experiments. (I) Results of pull-down assay as visualized by western blot. n = 3 independent experiments. (J) Immunofluorescence staining was employed to assess the nuclear translocation of ZNF460 in HL-60 neutrophils with different treatments. Scale bar, 4 μm; n = 3 independent experiments. (K) Immunofluorescence staining was employed to assess the expression of GGT1 in HL-60 neutrophils with different treatments. Scale bar, 4 μm; n = 3 independent experiments. (L) After the cells underwent indicated treatments, GGT1 expression was detected. n = 3 independent experiments. (M) After the cells underwent indicated treatments, taurine content was detected. n = 3 independent experiments. (N) mRNA levels of cdo , csad , fmo1 , and baat as measured using RT-qPCR. n = 3 independent experiments. Data were shown as mean ± standard deviation (SD) from biological replicates, and statistical comparisons were performed using unpaired Student’s t test in (D, G, I, and N) and one-way ANOVA followed by Tukey’s multiple comparisons test in (D, E, H, and J–M). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Taq Pro U + multiple probe qPCR mix kit , Vazyme , Cat#QN213-01.

Techniques: Binding Assay, Luciferase, Reporter Gene Assay, Thermal Shift Assay, Western Blot, Pull Down Assay, Immunofluorescence, Staining, Translocation Assay, Expressing, Quantitative RT-PCR, Standard Deviation