Journal: Bone Research

Article Title: Piezo1 channel activation in response to mechanobiological acoustic radiation force in osteoblastic cells

doi: 10.1038/s41413-020-00124-y

Figure Lengend Snippet: Fluorescence imaging of calcium oscillation and the effects of LIPUS stimulation on different groups of cells. a After LIPUS stimulation, the Fluo-8-labeled cells exhibited increased intracellular calcium levels at different time points. The red arrows show the two-cell calcium oscillation phenomenon. Scale bars, 50 μm. b Representative intracellular calcium traces of three groups of cells (MC3T3-E1, shRNA-Piezo1, and GsMTx4-treated cells) are shown as the fold increase in Fluo-8 intensity in response to LIPUS stimulation. The experiment was performed for six total minutes, including 1 min of baseline without LIPUS stimulation, 3 min of active stimulation (between the two red lines), and 2 min of regression. Time-lapse sequences were collected every 1.8 s for 6 min. In each field of interest, the fluorescence intensities of 10 cells were quantified using LSM Image Browser software

Article Snippet: In the GsMTx4 inhibition experiments, 1.5 mL of α-MEM with GsMTx4 (TOCRIS, MN, USA) at a concentration of 4 μmol·L −1 was added to the dishes.

Techniques: Fluorescence, Imaging, Labeling, shRNA, Software

Journal: iScience

Article Title: Astrocytes sense glymphatic-level shear stress through the interaction of sphingosine-1-phosphate with Piezo1

doi: 10.1016/j.isci.2024.110069

Figure Lengend Snippet: List of the chemical compounds studied

Article Snippet: GsMTx4 , Tocris , 4912.

Techniques: Concentration Assay

Journal: iScience

Article Title: Astrocytes sense glymphatic-level shear stress through the interaction of sphingosine-1-phosphate with Piezo1

doi: 10.1016/j.isci.2024.110069

Figure Lengend Snippet: PIEZO1 determines the mechanosensitivity of astrocytes (A) Representative experiment showing that GsMTx4 (1 μM), a PIEZO1 antagonist, reversibly inhibits the astrocyte calcium responses to flow. 5.8 dyn/cm 2 shear flow for 10 s (thin arrows) was applied at 300-s intervals before, after drug additions and after washout. Wash-in and washout of drug (thick arrows) were done at 0.2 mL/min for 2 min. (B) Summary of 3 independent experiments showing the reduction of shear-induced response by 1 μM GsMTX4 (red bar) and partial recovery (gray bar) upon washout. One-way ANOVA followed by Tukey’s post hoc test: ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. (C and D) Representative experiments showing the amplification of astrocyte calcium shear response by Yoda1, a PIEZO1 activator. A descending series of 10-s shear forces was applied in flow medium containing 0 or 45 μM BSA (C and D, respectively), then 10 μM Yoda1 was added and an ascending series of 10-s shear forces was applied. Shear forces (dyn/cm 2 ) are indicated above each response. Note: substantially enhanced responses in the presence of Yoda1. (E) Summary data comparing Yoda1 effect in various BSA concentrations (0, 7.5, and 45 μM) at moderate shear forces. Note: significant enhancement of sensitivity in the presence of Yoda1 at all BSA concentrations. Two-way mixed ANOVA: 45 μM BSA with vs. without Yoda: p value = 0.0024; F(1,13) = 14.15; 7.5 μM BSA with vs. without Yoda: p value = 0.0021; F(1,21) = 12.28; 0 μM BSA with vs. without Yoda: p value = 0.0202; F(4,28) = 3.468. (F) Representative experiment of calcium response of astrocytes incubated in FTY720 for 1.5 h. A descending series of 10-s shear forces was applied in flow medium containing 45 μM BSA, then 10 μM Yoda1 was added (bold black arrow) and an ascending series of shear forces was applied; open arrowhead indicates refocusing. (G) Summary of 3 independent experiments showing the block of flow-induced stress after FTY720 incubation and the recovery of response upon the addition of Yoda1. Unpaired Student’s t test: ∗ p < 0.05; ∗∗ p < 0.01. n ≥ 3 for all experiments. All data shown as mean ± SEM.

Article Snippet: GsMTx4 , Tocris , 4912.

Techniques: Shear, Amplification, Incubation, Blocking Assay

Journal: iScience

Article Title: Astrocytes sense glymphatic-level shear stress through the interaction of sphingosine-1-phosphate with Piezo1

doi: 10.1016/j.isci.2024.110069

Figure Lengend Snippet:

Article Snippet: GsMTx4 , Tocris , 4912.

Techniques: Virus, Recombinant, Saline, Modification, Software, Cell Culture, Fluorescence, Microscopy