Journal: Molecular Therapy

Article Title: Targeting Rap1b signaling cascades with CDNF: Mitigating platelet activation, plasma oxylipins and reperfusion injury in stroke

doi: 10.1016/j.ymthe.2024.09.005

Figure Lengend Snippet: PRP derived from hemorrhagic stroke patients exhibits lower levels of CDNF, but higher aggregation responses compared with healthy controls Extracellularly added CDNF suppressed certain agonist-induced platelet aggregation in PRP. (A) A flow diagram illustrates the experimental design process used in this study. (B) CDNF concentrations in PRP were measured using ELISA in healthy controls, traumatic hemorrhage, and hemorrhagic stroke patients. ∗∗∗ p < 0.0001 in comparison with the health donor. Data were analyzed by one-way ANOVA followed by Bonferroni corrections. (C–E) Representative the aggregation rates of healthy donor, traumatic hemorrhagic, and stroke patients’ PRP stimulated with various doses of ADP (C), TRAP6 (D), or collagen (E). Data were analyzed one-way ANOVA + post hoc Bonferroni test, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.0001 in comparison with the healthy controls. (F and G) Changes in platelet glycoprotein VI (GPVI) expression were observed in PRP obtained from healthy donors or stroke patients by flow cytometry. (G) Quantification of GPVI + /CD61 + expression in PRP ( n > 6, each group) were analyzed as two-tailed Student’s t test. ∗ p < 0.05 in comparison with the healthy donors. (H–K) Aggregation responses of PRP obtained from healthy controls and patients with traumatic brain hemorrhage or hemorrhagic stroke treated with different agonists. Collagen- (J)- and AA- (K) induced stroke patient’s PRP aggregation were significantly suppressed by CDNF treatment in an agonist dose-dependent manner. One-way ANOVA + original FDR method of Benjamini and Hochberg, ∗ p < 0.05 in comparison with the PBS group. Mean ± SEM is shown.

Article Snippet: The platelet samples were then labeled with anti-P-selectin-PE (1:100 for human sample– BD Biosciences), anti-TMRM-PE (1:100 for human sample– BD Invitrogen), anti-CD61-PerCP (1:150 for human sample– BD Biosciences), anti-P-selectin-PE (1:100 for animal sample– Bio-Rad), or anti-CD61-FITC (1:100 for animal sample– Invitrogen) for 30 min in the dark and fixed with 0.1% formaldehyde.

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Comparison, Expressing, Flow Cytometry, Two Tailed Test

Journal: Molecular Therapy

Article Title: Targeting Rap1b signaling cascades with CDNF: Mitigating platelet activation, plasma oxylipins and reperfusion injury in stroke

doi: 10.1016/j.ymthe.2024.09.005

Figure Lengend Snippet: Post-stroke systemic administration of CDNF reduced GPVI expression in PRP and downregulated ERK-cPLA 2 -TXA 2 signaling transduction in circulating platelets (A) Timetable of experiment. The rats underwent dMCAo surgery, and they were divided into two groups randomly. Fifteen minutes after reperfusion, the animals received an s.c. injection of either CDNF or PBS (vehicle). PRP or washed platelet samples were collected at 6 h and 1 day post-surgery. Behavioral functions were evaluated on days 2, 7, and 14 post-stroke. Rats were sacrificed for analysis at different time points. (B and C) By using flow cytometry, it was observed that CDNF treatment suppressed the elevated expression of GPVI + /CD61 + in PRP at 6 h after dMCAo. (C) Quantitation of GPVI + /CD61 + expression in PRP derived from naive, CDNF alone, dMCAo + vehicle and dMCAo + CDNF groups. n = 5–6 per group. ∗∗∗ p < 0.001 by Tukey’s multiple comparisons test, following one-way ANOVA. (D–F) Western blot analysis revealed that systemic administration of CDNF suppressed the upregulated expressions of p-ERK (E) and p-cPLA 2 (F) in washed platelets at 6 h post-stroke. cPLA 2 or ERK was used as an internal control for normalization. The statistical analysis of the results is presented as a fold change relative to the naive group. ∗∗ p < 0.01, ∗∗∗ p < 0.001 indicates comparison with the dMCAo + vehicle group by Dunnett’s multiple comparisons test, following one-way ANOVA. (G–I) The activity of p-cPLA 2 as well as production of TXB 2 and 12-HETE in washed platelets at 6 h after dMCAo were measured using ELISA. The findings revealed that CDNF treatment suppressed stroke-upregulated cPLA 2 activity (G) and the production of TXB 2 by platelets (H), while it had no effect on the synthesis of platelet 12-HETE (I). ∗ p < 0.05, ∗∗ p < 0.001, Holm-Šidák multiple comparisons test, following one-way ANOVA. The mean ± SEM of three independent experiments is shown.

Article Snippet: The platelet samples were then labeled with anti-P-selectin-PE (1:100 for human sample– BD Biosciences), anti-TMRM-PE (1:100 for human sample– BD Invitrogen), anti-CD61-PerCP (1:150 for human sample– BD Biosciences), anti-P-selectin-PE (1:100 for animal sample– Bio-Rad), or anti-CD61-FITC (1:100 for animal sample– Invitrogen) for 30 min in the dark and fixed with 0.1% formaldehyde.

Techniques: Expressing, Transduction, Injection, Flow Cytometry, Quantitation Assay, Derivative Assay, Western Blot, Control, Comparison, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: Biomarker Research

Article Title: The involvement of krüppel-like transcription factor 2 in megakaryocytic differentiation induction by phorbol 12-myrestrat 13-acetate

doi: 10.1186/s40364-024-00614-9

Figure Lengend Snippet: Krüppel-like factor 2 (KLF2) regulates the expression of megakaryocytic markers CD61 and CD41. The lentiviral vectors harboring pLVX-IRES-Puro encoding KLF2 (lenti-pLVX-KLF2), two pLVX-shRNAs against KLF2 (lenti-pLVX-shKLF2 #1 / #2 ), and their control vectors were respectively constructed. The lentiviral particles were infected into K562 or HEL cells for 72 h. Subsequently, the cells were treated with 25 nM phorbol 12-myristate 13-acetate (PMA) and further incubated for 2, 4, and 6 days. Representative contour plots were used to identify CD41 + /CD61 + cells in K562 ( A ) cells and HEL ( B ) cells

Article Snippet: Then the cells were collected, washed with phosphate balanced solution (PBS), and stained with PE Anti-Human CD41 (Thermo Fisher Scientific Inc., Pittsburgh, PA, USA) and FITC Anti-Human CD61 (Proteintech Group, Inc., Rosemont, IL, USA).

Techniques: Expressing, Control, Construct, Infection, Incubation

Journal: Biomarker Research

Article Title: The involvement of krüppel-like transcription factor 2 in megakaryocytic differentiation induction by phorbol 12-myrestrat 13-acetate

doi: 10.1186/s40364-024-00614-9

Figure Lengend Snippet: Chimerin 1 (CHN1) and potassium voltage-gated channel subfamily Q member 5 (KCNQ5) may influence megakaryocyte differentiation. siRNAs targeting CHN1 or KCNQ5 were synthesized separately and then transfected into K562 cells along with their respective control siRNAs. Twenty-four hours after transfection, the cells were treated with 25 nM phorbol 12-myristate 13-acetate (PMA) for 6 days. Representative contour plots were used to identify CD41 + /CD61 + cells in the CHN1 siRNA or KCNQ5 siRNA transfected cells, respectively

Article Snippet: Then the cells were collected, washed with phosphate balanced solution (PBS), and stained with PE Anti-Human CD41 (Thermo Fisher Scientific Inc., Pittsburgh, PA, USA) and FITC Anti-Human CD61 (Proteintech Group, Inc., Rosemont, IL, USA).

Techniques: Synthesized, Transfection, Control