Journal: Cell Communication and Signaling : CCS

Article Title: Head and neck squamous cell carcinoma-derived extracellular vesicles mediate Ca²⁺-dependent platelet activation and aggregation through tissue factor

doi: 10.1186/s12964-025-02215-x

Figure Lengend Snippet: EV-induced PLT aggregation and activation involve the coagulation cascade. PLTs were suspended in Tyrode’s buffer containing 2 mM Ca 2+ , and aggregation was monitored for 1000 s. (A) Schematic overview of used PLT aggregation inhibitors and their targets within the PLT activation and aggregation pathways. This graph was created using BioRender.com. (B) Role of phospholipase C (PLC) in EV-induced PLT aggregation. PLTs were pre-incubated with U73122 (10 µM; +; PLC inhibitor), without U73122 (-) or Dimethyl sulfoxide (DMSO; 1:250 dilution; ctrl) for 300 s, followed by the addition of SAS- or UD5 derived EVs (60 µg/mL). PLT aggregation was quantified as the area under the curve (AUC, n = 4–8). (C) Effect of FcγRIIa inhibition on PLT aggregation. PLTs were pre-incubated with (+) or without (-) the FcγRIIa-blocking antibody IV.3 (300 ng/mL) for 300 s in an aggregometer. IgG-coated E. coli (5 × 10⁷ bacteria/sample) or SAS and UD5 EVs (60 µg/mL) were then added to assess FcγRIIa-dependent PLT activation ( n = 3–9). (D) Involvement of PAR1 and PAR4 thrombin receptors in EV-induced PLT aggregation. PLTs were pre-treated with Vorapaxar (+; Vora.; 10 µM; PAR1 inhibitor) and BMS986120 (+; BMS; 10 µM; PAR4 inhibitor) or vehicle control (-; DMSO, 1:250 dilution) for 300 s, followed by the addition of SAS or UD5-derived EVs (60 µg/mL). Thrombin receptor activation was induced using TRAP (25 µM; thrombin receptor agonist) as a positive control ( n = 5–11). (E) PLTs were treated with the inhibitor PPACK (+, 1 µM) or DMSO control (-, 1:20,000 dilution) to block thrombin activity, and aggregation was assessed following stimulation with SAS and UD5 EVs (60 µg/mL) or TRAP (25 µM; positive control) ( n = 5–10). (F) PLT aggregation following treatment with the thrombin inhibitor hirudin (+, 2.5 µM) or H 2 O control (-, 1:200 dilution) was measured after stimulation with SAS and UD5 EVs (60 µg/mL) or TRAP (25 µM) ( n = 3–8). (G) Thrombin activity during PLT aggregation. Thrombin activity (IU/mL) was measured at 10 s, the start of aggregation, and at 50% aggregation. PLTs were incubated with SAS- or UD5-derived EVs (60 µg/mL), and supernatants were collected for analysis. PBS-treated PLTs served as controls ( n = 5, left graph; n = 4, right graph). Data are presented as the mean + SD from n ≥ 3 independent experiments. Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Kruskal-Wallis test followed by Dunn’s post-hoc test for (B-D). One-way ANOVA followed by Bonferroni’s post-hoc test for (E, F). Two-way ANOVA followed by Tukey’s post-hoc test for (G)

Article Snippet: Inhibitors used included U73122 (#J62898.MCR, ThermoFisher Scientific), Phe-Pro-Arg-Chloromethylketone (PPACK, #sc-201291, Santa Cruz Biotechnology), Hirudin (#H0393-100UN, Merck), Vorapaxar (#23119, Cayman Chemical), and BMS986120 (#23497-01, Cayman Chemical).

Techniques: Activation Assay, Coagulation, Incubation, Derivative Assay, Inhibition, Blocking Assay, Bacteria, Control, Positive Control, Activity Assay

Journal: FEBS Open Bio

Article Title: PLCγ1 inhibition combined with inhibition of apoptosis and necroptosis increases cartilage matrix synthesis in IL‐1β‐treated rat chondrocytes

doi: 10.1002/2211-5463.13064

Figure Lengend Snippet: U73122‐induced apoptosis and necroptosis in IL‐1β‐treated rat chondrocytes, and the effect of combined treatment with the apoptosis and necroptosis inhibitors Z‐VAD and Nec‐1, respectively. Rat chondrocytes pretreated with IL‐1β (20 ng·mL −1 for 36 h) were treated with U73122 (2 μ m for 12 h). Cell proliferation (A), percentage of dead cells (B), and ROS level (C) were measured. Protein levels of apoptosis and necroptosis indexes Bcl‐2, Bax, P53, pro/cleaved‐caspase3, RIP1, and RIP3 were analyzed by Western blotting (D). Chondrocytes pretreated by IL‐1β (20 ng·mL −1 for 36 h) and U73122 (2 μ m for 12 h) were treated with Z‐VAD (10 μ m for 12 h) or/and Nec‐1 (30 μ m for 12 h). Cell proliferation (E), percentage of dead cells (F), and ROS level (G) were measured, and protein levels of apoptosis and necroptosis indexes were analyzed by Western blotting (H). The results of ROS were normalized, means of the first group in Fig. were used as the control for ROS. β‐Actin was used as the control for Western blotting. Values are means and standard deviations, the error bars represent SD. One‐way ANOVA with the Dunnett test was used to calculate P values. These results are representative of at least three independent experiments in each experiment. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Inhibitors used in this study (U73122, Z‐VAD, and Nec‐1) were purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Western Blot, Control

Journal: FEBS Open Bio

Article Title: PLCγ1 inhibition combined with inhibition of apoptosis and necroptosis increases cartilage matrix synthesis in IL‐1β‐treated rat chondrocytes

doi: 10.1002/2211-5463.13064

Figure Lengend Snippet: U73122 combined with apoptosis and necroptosis inhibitors increased Collagen2 and Aggrecan levels in IL‐1β‐treated rat chondrocytes. Chondrocytes pretreated by IL‐1β (20 ng·mL −1 for 36 h) and U73122 (2 μ m for 12 h) were treated with Z‐VAD (10 μ m for 12 h) or/and Nec‐1 (30 μ m for 12 h). Protein (A) and mRNA (B) levels of Collagen2 and Aggrecan were analyzed by Western blotting and RT‐PCR, respectively. β‐Actin was used as the control for Western blotting, and GAPDH was used as the control for RT‐PCR. Values are means and standard deviations, the error bars represent SD. One‐way ANOVA with the Dunnett test was used to calculate P values. These results are representative of at least three independent experiments in each experiment. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Inhibitors used in this study (U73122, Z‐VAD, and Nec‐1) were purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Control

Journal:

Article Title: Involvement of Src tyrosine kinase and mitogen-activated protein kinase in the facilitation of calcium channels in rat nucleus of the tractus solitarius by angiotensin II

doi: 10.1113/jphysiol.2005.095307

Figure Lengend Snippet: A, typical superimposed ICa traces at the times indicated in the time course of graph B. B, typical time course of Ang II-induced facilitation of ICa in a neuron treated with PLC inhibitor U-73122 (10 μm for 15 min before patch clamp experiments). C, histogram demonstrating the degree of ICa facilitation by 1 μm Ang II in control (untreated neurons), after 5, 10, 20 and 50 μm U-73122. Numbers in parentheses indicate the number of neurons tested. D, typical superimposed ICa traces at the times indicated in the time course graph E. E, typical time course of Ang II-induced facilitation of ICa in a neuron treated with PI3K inhibitor LY294002 (10 μm for 10 min before patch clamp experiments). Ang II (1 μm) was bath-applied during the time indicated by the filled bar.

Article Snippet: U-73122 was purchased from Wako Pure Chemical Industries (Osaka, Japan).

Techniques: Patch Clamp

Journal:

Article Title: Involvement of Src tyrosine kinase and mitogen-activated protein kinase in the facilitation of calcium channels in rat nucleus of the tractus solitarius by angiotensin II

doi: 10.1113/jphysiol.2005.095307

Figure Lengend Snippet: A, comparison of ICa values in various conditions in the presence of DMSO in the pipette solution. Histogram demonstrating the degree of ICa values before application of any drugs, application of 1 μm Ang II, genistein alone in the pipette solution, genistein in the pipette solution + 1 μm Ang II, lavendustin A alone in the pipette solution, lavendustin A in the pipette solution + 1 μm Ang II, PD98,059 alone in the pipette solution and PD98,059 in the pipette solution + 1 μm Ang II. B, histogram demonstrating the degree of ICa facilitation by 1 μm Ang II in control (untreated neurons), after U-73122, LY294002, genistein, lavendustin A, PP2, PD98,059, U0126, SB202190 and SP600125. Numbers in parentheses indicate the number of neurons tested. *P < 0.05 compared with control, ANOVA.

Article Snippet: U-73122 was purchased from Wako Pure Chemical Industries (Osaka, Japan).

Techniques: Transferring