Journal: Research and Practice in Thrombosis and Haemostasis

Article Title: Carbon dioxide alleviates platelet storage lesions via stimulating fatty acid metabolism and reducing platelet glucose consumption

doi: 10.1016/j.rpth.2025.102681

Figure Lengend Snippet: Carbon dioxide (CO 2 ) inhibited platelet activation and apoptosis during storage. (A) The percentage of CD62P-positive platelets was determined by flow cytometry. (B) Platelet factor 4 (PF4) concentration was determined by enzyme-linked immunosorbent assay. (C) The percentage of annexin V-positive platelets was determined by flow cytometry. (D) The mean fluorescence intensity (MFI) of CD42b was determined by flow cytometry ( n = 6). n.s., not significant, ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001, ∗∗∗∗ P < .0001.

Article Snippet: To determine PF4 concentration, 200 μL of supernatant from stored APCs (300 μL, 1 × 10 9 /mL) was processed following the PF4 ELISA Kit instructions (Boster).

Techniques: Activation Assay, Flow Cytometry, Concentration Assay, Enzyme-linked Immunosorbent Assay, Fluorescence

Journal: Journal of Neuroinflammation

Article Title: Induction of neurodegeneration in the hippocampus of senescence-accelerated mouse-prone 8 (SAMP8) mice by blood-brain barrier-crossing serum amyloid P component

doi: 10.1186/s12974-026-03704-7

Figure Lengend Snippet: Blood-derived SAP promotes neuronal apoptosis and synaptic density decline in a primary cortical neuron culture. ( A ) Schematic diagram showing the study design to quantify apoptosis and synaptic density in E15.5 mouse-derived primary cortical neurons treated with the sera of SAMR1 and SAMP8 mice for 48 h, recombinant SAP for 6 h, and the neutralizing anti-SAP antibody for 2 h before sera treatment. ( B ) Confocal images of cleaved-caspase 3 (red) and MAP2 (green) stained cortical neurons treated with vehicle, 1000 µg/mL total serum protein of SAMR1 and SAMP8 mice, 60 nM recombinant SAP, or 500 µg/mL total serum protein of SAMP8 mice and the neutralizing anti-SAP antibody, showing that apoptotic cells with cleaved-caspase 3 + granules (arrowheads). Cleaved-caspase 3; MAP2 double positive neurons (arrows) are counted as cleaved-caspase 3 + neurons. ( C ) Ratio of cleaved-caspase 3 + apoptotic neurons in primary cortical neurons treated with 100, 250, 500, or 1000 µg/mL serum protein of SAMR1 and SAMP8 mice. Data represent mean ± SEM from at least three independent cultures. Statistic uses two-way ANOVA. ( D ) ELISA for SAP concentration in the sera of 6-month-old SAMR1 and SAMP8 mice. The SAP volume is normalized with fluid volume of sera. The dashed line indicates the median, dot lines indicate quartiles, and dots indicate the results of each mouse. Statistic uses unpaired and two-tailed t test. ( E ) Ratio of cleaved-caspase 3 + apoptotic neurons in primary cortical neuron cultures treated with vehicle and 60 nM recombinant SAP. Data represent mean ± SEM from four independent cultures, and dots indicate the results from each experimental culture. Statistic uses unpaired and two-tailed t test. ( F ) Ratio of cleaved-caspase 3 + apoptotic neurons in primary cortical neurons treated with control rabbit IgG and neutralizing anti-SAP antibody 2 h before the treatment with 500 µg/mL serum protein of SAMR1 and SAMP8 mice. Data represent mean ± SEM from four independent cultures, and dots indicate the results from each experimental culture. Statistic uses two-way ANOVA. ( G ) Confocal images of synaptophysin (magenta) and MAP2 (green)-, and PSD95 (magenta) and MAP2 (green)-stained cortical neurons treated with vehicle, 500 µg/mL serum protein of SAMR1 and SAMP8 mice, 60 nM recombinant SAP, or 500 µg/mL serum protein of SAMP8 mice and the neutralizing anti-SAP antibody. ( H , I ) Colocalized area of Synaptophysin + presynapses or PSD95 + postsynapses with MAP2 + cytoplasmic and neurite area in primary cortical neurons treated with vehicle and 500 µg/mL serum protein of SAMR1 and SAMP8 mice. Data represent mean ± SEM from four independent cultures. Statistics use two-way ANOVA. ( J , K ) Colocalized area of Synaptophysin + presynapses or PSD95 + postsynapses with MAP2 + neuronal area in primary cortical neurons treated with vehicle and 60 nM recombinant SAP. Data represent mean ± SEM from five independent cultures, and dots indicate the results from each experimental culture. Statistic uses unpaired and two-tailed t test. ( L , M ) Colocalized area of Synaptophysin + presynapses or PSD95 + postsynapses with MAP2 + neuronal area in primary cortical neurons treated with control rabbit IgG and neutralizing anti-SAP antibody 2 h before the treatment with 500 µg/mL sera of SAMR1 and SAMP8 mice. Data represent mean ± SEM from four independent cultures, and dots indicate the results from each experimental culture. Statistics uses two-way ANOVA. ns, not significant

Article Snippet: Sera from control SAMR1 and SAMP8 mice were added to culture media at final total serum protein concentrations of 100, 250, 500, or 1,000 μg /mL for 48 h. Recombinant mouse SAP (50113-M08H, Sino Biological, Kawasaki, Japan) was added at a final concentration of 60 nM for 6 h. PBS served as the vehicle for both recombinant SAP and serum.

Techniques: Derivative Assay, Recombinant, Staining, Enzyme-linked Immunosorbent Assay, Concentration Assay, Two Tailed Test, Control

Journal: The Journal of Biological Chemistry

Article Title: RUNX3 Maintains the Mesenchymal Phenotype after Termination of the Notch Signal *

doi: 10.1074/jbc.M111.222331

Figure Lengend Snippet: RUNX3 is a direct target gene of Notch in human endothelial cells and mouse embryonic hearts. A, Dll4 or Jag1 coculture was performed using HMEC transduced with FLAG-CSL-R179H or vector. Expression of RUNX3 and CSL-R179H in the whole cell lysates were examined by immunoblotting. Tubulin was used as a loading control. B, HMEC were transduced with dnMAML, NICD, or both. Expression of RUNX3 in the whole cell lysates was examined by immunoblotting. Tubulin was used as a loading control. C, HMEC were transduced with FLAG-CSL or vector. Expression of FLAG-CSL and endogenous CSL in whole cell lysates were examined by immunoblotting using anti-FLAG and anti-CSL antibodies, respectively. Tubulin was used as a loading control. D, CSL occupancy on CSL binding sites in the RUNX3 or SMA promoter was examined by ChIP using anti-FLAG antibody with IgG as negative control. ChIP-qPCR was conducted using primers flanking CSL sites in RUNX3 and SMA promoters. The occupancy of CSL on these sites were calculated as percentage of the respective input DNA concentration and expressed as relative signal after normalized against the vector antibody samples (designated as 1). Values are shown as mean ± S.E. of four independent experiments. *, significantly higher (p < 0.05) than all other three samples. E, CSL occupancy on CSL binding sites in the Runx3 and SMA promoters in mouse embryonic hearts was examined by ChIP using anti-CSL antibody with IgG as negative control. ChIP-qPCR was conducted using primers flanking CSL sites on the mouse Runx3 and SMA promoters. The occupancy of CSL on these sites were calculated as percentage of the respective input DNA concentration and expressed as relative signal after normalized against the IgG samples (designated as 1). Values are shown as mean ± S.E. of four independent experiments. *, significantly higher (p < 0.05) than the respective IgG samples.

Article Snippet: After the cross-linking and sonication, equal amount of chromatin (15 μg) was used for ChIP assay using 1 μg CSL antibody (ProteinTech Group, Inc.) or 1 μg normal rabbit IgG (Vector Laboratories, Burlingame, CA) as negative control.

Techniques: Transduction, Plasmid Preparation, Expressing, Western Blot, Binding Assay, Negative Control, Concentration Assay