Journal: The Journal of Experimental Medicine

Article Title: Filamin A editing in myeloid cells reduces intestinal inflammation and protects from colitis

doi: 10.1084/jem.20240109

Figure Lengend Snippet: A fixed, fully FLNA R in myeloid cells shifts cellular properties and could be therapeutically exploited in colitis. (A) Flna mRNA editing status of WT whole distal colon was determined by RT-PCR amplicon Illumina sequencing on days (d) 0, 2, 5, and 10 of DSS challenge (one-way ANOVA, F = 19.09, DF = 3). (B) Gene expression analysis by real-time qPCR from WT colon homogenates. CT values relative to Gapdh (2^−ddCT) are shown as log 2 fold change compared with day zero depicted as heatmap at different time points of DSS treatment (mixed-effects analysis followed by Tukey’s multiple comparisons test). (C–F) Flna mRNA editing status of sorted cell populations of two–six pooled WT mouse colons, BM, and blood determined by RT-PCR amplicon Illumina sequencing on days 0, 2, 5, and 10 of DSS challenge (mixed-effects analysis followed by Tukey’s multiple comparisons test). (G and H) Bar graphs of cytokine levels (pg/ml) in supernatants from LPS-stimulated mouse neutrophils and BMDMs (Student’s t test). (I) Neutrophil migration speed in a pillar forest of a microfabricated PDMS device toward an fMLP gradient (Student’s t test). Dashed lines represent the median. (J) Exemplary image of CSFE- and TAMRA-stained FLNA Q and FLNA R neutrophils within the device. (K and L) Quantification of DNA release as relative fluorescence units to measure NETosis of isolated neutrophils after stimulation with a23187 or PMA over time. Time course data were plotted in a line graph with mean ± SEM (two-way ANOVA followed by Tukey’s multiple comparisons test, F = 11.94, DF = 60). (M and N) Scoring of histological colon sections stained for NETs by anti-citrullinated histone 4 (citH4) or neutrophils by anti-mouse Ly-6G. (O) Flna editing levels in intestinal tissues of WT mice: healthy mice, mice with acute colitis (day 10 of DSS), or mice recovered from colitis (day 21) were analyzed for Flna editing levels in distal and proximal colon separately ( n = 4 per time point; one-way ANOVA, F = 9.208, DF = 18). (P) FLNA editing levels in human colon biopsies of healthy donors ( n = 9), patients suffering from active UC ( n = 6), and patients in remission from active UC ( n = 6). Distal and proximal colon biopsies were analyzed separately (one-way ANOVA, distal: F = 7.326, DF = 15); comparison healthy-distal versus healthy-proximal (Student’s t test). ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05. Error bars show SEM. Data in A–F are pooled from two independent experiments ( n = 3–5 pooled colons per group, per experiment). Data in G–L are representative of two experiments, data in M and N are pooled from two experiments, and data in O are from a single experiment.

Article Snippet: a23187 , Merck/MilliporeSigma , Cat#C7522.

Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, Illumina Sequencing, Gene Expression, Migration, Staining, Fluorescence, Isolation, Comparison

Journal: Advanced Science

Article Title: Succinate Dehydrogenase Subunit A (SDHA) Mediated Microglia Extracellular Traps Formation Participating in Cerebral Ischemic Reperfusion Injury

doi: 10.1002/advs.202411873

Figure Lengend Snippet: OGD/R induced MiETosis in vitro. A) Cell viability of micoglia was detected by CCK‐8 assay after OGD (4 h) following re‐oxygenation for 6, 12, 24 h, and B) incubation with A23187 (5 µM) for 1, 2, 4, 6 h. C) Representative images of microglia following incubation with the Hoechst 33342 show the presence of nuclear decondensation at 40 × magnification. Scale bar = 10 µm. D) Quantification of percentage of microglia undergoing nuclear decondensation. E) Representative images of Iba1, citH3 and MPO triple‐stained microglia, and colocalization analysis of MPO, citH3 and DNA in three group. Scale bar = 50 µm F,G) Quantitative analysis of MPO and citH3 mean immunofluorescence intensities (MFI) in each group of microglia. H) MPO, I) ds‐DNA) concentrations in microglia culture supernatant, as detected by ELISA. J) Scanning electron microscopy (SEM) results demonstrated that microglia released porous, protein‐rich extracellular structures following OGD/R treatment. K,L) Representative immunoblot and statistical analysis of citH3, MPO, MMP9, PAD2, PAD4 and β‐actin after OGD/R exposure. M) Representative images of microglia following incubation with the fluorescent probe mitoSOX show the production of mtROS at 40 × magnification. Scale bar = 10 µm. N) Single‐color histogram of mitoSOX fluorescence intensity. All images are representative and were chosen at random. O) Quantification of mean intensities of mitoSOX. All experiments were performed at least three independent times. All data are presented as mean ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: A23187 (10 mM, 52665‐69‐7; MedChemExpress) stock solutions were dissolved in DMSO at a storage concentration.

Techniques: In Vitro, CCK-8 Assay, Incubation, Staining, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Electron Microscopy, Western Blot, Fluorescence

Journal: Advanced Science

Article Title: Succinate Dehydrogenase Subunit A (SDHA) Mediated Microglia Extracellular Traps Formation Participating in Cerebral Ischemic Reperfusion Injury

doi: 10.1002/advs.202411873

Figure Lengend Snippet: Inhibition of MiETs formation protected neuron from injury in vitro. A,E) Schematic illustration of the experimental design framework for this section. B) Cell viability of neurons was detected by CCK‐8 assay after incubation with supernatant from microglia in Con, A23187 and OGD/R groups. C) PI‐Hoechst double‐stained neurons treated with four types of microglia culture medium. D) Quantification of PI/Hoechst double‐positive cells. Scale bar = 50 µm. F) Cell viability of neurons was detected by CCK‐8 assay after incubation with supernatant from microglia in Con, OGD/R, BB‐Cl, and DMM groups. G) PI‐Hoechst double‐stained neurons treated with five types of microglia culture medium. H) Quantification of PI/Hoechst double‐positive cells in these five groups. Scale bar = 50 µm. All experiments were performed at least three independent times. All data are presented as mean ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: A23187 (10 mM, 52665‐69‐7; MedChemExpress) stock solutions were dissolved in DMSO at a storage concentration.

Techniques: Inhibition, In Vitro, CCK-8 Assay, Incubation, Staining

Journal: Journal of Inflammation Research

Article Title: Feedback Regulation of sPLA2-COX/5-LOX-Ca2+ in Seminal Plasma and Its Impact on Sperm Quality Parameters

doi: 10.2147/JIR.S523172

Figure Lengend Snippet: Impact of in vitro Addition of 100 pg AA + 300 pg COX1 on Seminal Plasma AA Metabolic Network Indicators Under 42°C Heat Stress

Article Snippet: Calcium ion carrier A23187, anti-CD46-FITC fluorescent dye staining, and PI fluorescent dye [Celula (China) Medical Technology Co., Ltd., Sichuan] were utilized to assess sperm viability and acrosome reaction capacity.

Techniques: In Vitro, Clinical Proteomics, Control