Journal: Nature Communications

Article Title: Highly specific and non-invasive imaging of Piezo1-dependent activity across scales using GenEPi

doi: 10.1038/s41467-023-40134-y

Figure Lengend Snippet: a Representative example of GenEPi activation and b F/F 0 signal intensity profile (black) in response to 10 dyne/cm 2 shear stress (green) in HEK293T cells. Time stamps in the images correspond to the stimulation and response profile in the graph. Scale bar, 10 μm. c Response of HEK293T cells expressing Piezo1 and GCaMP-G4 ( n = 13, shear stress; n = 18, ionomycin), GenEPi ( n = 12, shear stress; n = 15, ionomycin), or Piezo1-eGFP ( n = 16, shear stress, n = 19, ionomycin) to shear stress and ionomycin. Two-tailed Mann–Whitney test, **** p < 0.0001; * p < 0.05; n.s. = p > 0.05, data from three independent experiments. Data were presented as means ± SEM. d Response of GenEPi-expressing HEK293T cells to 10 μM Yoda1 ( n = 14) or DMSO ( n = 17). Two-tailed Mann–Whitney test, **** p < 0.0001, data from three independent experiments. Data were presented as means ± SEM. e Response of GenEPi and jRCaMP1a expressing HEK293T cells ( n = 19) to intracellular Ca 2+ triggered by 30 μM ATP. Two-tailed unpaired t -test, **** p < 0.0001, data from six independent experiments. Data were presented as means ± SEM. f Representative images of AFM cantilever stimulation of GenEPi-expressing HEK293T cells stimulated by the compressing AFM cantilever. Brightfield image of cantilever position before (top left) and during stimulation (top right) and corresponding fluorescent images of the stimulated cell before and after stimulation. Scale bar, 10 μm. g The mechanical stimulation procedure of compressive forces ranging from 100–400 nN (purple) along with the brightfield (gray) and fluorescent (green) traces from the cell depicted in f . h Amplitude of Ca 2+ responses from GenEPi ( n = 21), and Piezo-eGFP ( n = 45) expressing cells. Data were presented as means ± SEM. Two-tailed Mann–Whitney test, **** p < 0.0001. i Amplitude of Ca 2+ responses of GenEPi in cells transfected with GenEPi before ( n = 13) and after addition of 3 μM GsMTx-4 ( n = 11). Data were presented as means ± SEM. Two-tailed Mann–Whitney test, **** p < 0.0001. j Threshold forces and pressures for cells co-transfected with human Piezo1 and cytosolic GCaMP-G4 ( n = 21), n.s. = p > 0.05, Data were presented as means ± SEM. Two-tailed unpaired t -test. k Duration of Ca 2+ responses from cells co-transfected with cytosolic GCaMP-G4 and human Piezo1 ( n = 27) and GenEPi ( n = 16). Two-tailed Mann–Whitney test, **** p < 0.0001. Data were presented as means ± SEM. Data from three independent experiments. l Representative traces of the currents from wild-type Piezo1 (black) and GenEPi (green) evoked by the negative pressure of 0, −20, −40, −60, −80, and −100 mmHg at −80 mV with the cell-attached recording configuration. The current evoked by individual negative pressure was normalized to the maximum evoked by the negative pressure of −100 mmHg. m Both wild-type Piezo1 ( n = 8 cells; 8 experiments) (black) and GenEPi ( n = 6 cells; 6 experiments) (green) show a similar pressure-dependent response. Data were presented as means ± SEM. Two-tailed unpaired t -test, p = 0.11, p = 0.06, p = 0.20, and p = 0.07 for pressure sensitivities to −20, −40, −60, and −80 mmHg between Piezo1 and GenEPi. n Representative traces of wild-type Piezo1 (black) or GenEPi (green) currents evoked by −60 mmHg pressure at different voltages of +30, −30, −50, and −80 mV. o The conductance value for wild-type Piezo1 (black) is 24.9 ± 0.8 pS ( n = 6 cells from six experiments, mean ± SEM) and the conductance value for GenEPi (green) is 25.5 ± 0.6 pS ( n = 6 cells from six experiments, mean ± SEM). Both channels show the typical 25 pS conductance of the Piezo1 channel, indicating that the ion selectivity of Piezo1 is preserved within GenEPi. p Example traces of wild-type Piezo1 channels (black) or GenEPi (green) activated by 0.1, 0.3, 1, 3, 10 µM Yoda1 in the absence of mechanical stimulation. After the channel activities were evoked by 10 µM Yoda1, 10 µM ruthenium red (RR) was used to inhibit the currents (blue). Source data are provided as a Source Data file.

Article Snippet: We used several concentrations of Yoda1 (5586, Tocris Bioscience) diluted in DMSO (D8418, Sigma-Aldrich) ranging from 5 to 50 μM and 2 mM EGTA (OmniPur EGTA, 4100) diluted in water.

Techniques: Activation Assay, Shear, Expressing, Two Tailed Test, MANN-WHITNEY, Transfection

Journal: Nature Communications

Article Title: Highly specific and non-invasive imaging of Piezo1-dependent activity across scales using GenEPi

doi: 10.1038/s41467-023-40134-y

Figure Lengend Snippet: a Schematic of the pTol2-hsp70:GenEPi plasmid used for the zebrafish transgenesis. GenEPi is downstream of the zebrafish hsp70-l promoter after the β-globin rabbit intron. b Schematic of the heat-shock protocol used to induce the expression of GenEPi in Tg(hsp70:GenEPi) zebrafish. c Longitudinal analysis of GenEPi expression upon heat-shock of Tg(hsp70:GenEPi) zebrafish at 1dpf, fluorescence steady state is reached after ~4–5 h. n = 35 fish. d Representative fluorescence of a non- (non-HS) and heat-shocked (HS) Tg(hsp70:GenEPi) zebrafish at 1 day post-fertilization (dpf). Scale bar, 200 μm. e Increase in fluorescence intensity following the heat-shock protocol in ( b ). ( n = 20 and n = 31 non-heat-shocked (non-HS) and heat-shocked (HS) fish, respectively). Data were presented as means ± SEM. f GenEPi expression in the developing eye, neural retina, lens fiber cells, and primary anterior epithelium in a 1dpf Tg(hsp70:GenEPi) zebrafish. Scale bar, 20, 200, and 20 μm, respectively. g Representative images of GenEPi expression in the eye of a 1dpf Tg(hsp70:GenEPi) zebrafish during the chemical modulation of channel’s activity by Yoda1 and RR. Scale bar, 50 μm. h Normalized fluorescence intensity of control and Tg(hsp70:GenEPi) zebrafish during the chemical modulation of GenEPi activity in 10 μM Yoda1, 10 μM Yoda1 and 10 μM RR, 10 μM RR, and wash out to E3 medium. Non-heat-shocked embryos which do not express GenEPi showed non-significant responses both in the presence or absence of Yoda1 or RR (paired data of n = 3 and n = 24 non-HS and HS transgenic zebrafish, respectively). Data were presented as means ± SEM. Two-way ANOVA test. i Representative image of a Tg(hsp70:GenEPi) zebrafish heart at 3dpf. GenEPi is expressed in both the endocardium (dashed line) and myocardium cells in the developing zebrafish heart. Scale bar, 20 μm. j Representative fluorescence intensity ( F/F 0 ) profile from the antrioventricular canal (AVC) of a Tg(hsp70:GenEPi) zebrafish heart which shows the two-phase GenEPi activation. k Representative fluorescence intensity ( F/F 0 ) profile from the same ROI in j at the AVC of the same Tg(hsp70:GenEPi) zebrafish heart after treatment with BDM which abolishes heart beating, showing absence of GenEPi responses. l Mean of 27 representative GenEPi responses during individual heart beating cycles at 1dpf. Normalization of the GenEPi signal was obtained by computing the ratiometric intensity of GenEPi to the average of 12 NLS-mCherry signals of individual heartbeats in Tg(kdrl:NLS-mCherry) zebrafish at 1dpf. Source data are provided as a Source Data file.

Article Snippet: We used several concentrations of Yoda1 (5586, Tocris Bioscience) diluted in DMSO (D8418, Sigma-Aldrich) ranging from 5 to 50 μM and 2 mM EGTA (OmniPur EGTA, 4100) diluted in water.

Techniques: Plasmid Preparation, Expressing, Fluorescence, Activity Assay, Control, Transgenic Assay, Activation Assay

Journal: JOR Spine

Article Title: Mechanosensitive Ion Channel PIEZO1 Suppresses BMP2 ‐Induced Ossification of the Annulus Fibrosus Cells

doi: 10.1002/jsp2.70168

Figure Lengend Snippet: Piezo1 mediates the suppression of osteogenic gene expression under low‐strain CTS. The knockdown efficiency was confirmed to be approximately 80% for each gene. (A) RT‐qPCR showed that si Piezo1 reversed the low‐strain CTS‐induced downregulation of Runx2 and Osx . (B, C) si Piezo2 or si Trpv4 did not reverse osteogenic gene suppression by low‐strain CTS. An ordinary one‐way analysis of variance (ANOVA), followed by Tukey's multiple comparisons test was used. Each group was compared with the siN/ C without the CTS group. Statistical significance was set at P < 0.05, with significance levels denoted as follows: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: We measured intracellular calcium responses using the calcium indicator dye Fluo‐8 NW (AAT Bioquest, Pleasanton, CA, Cat. #36315) and PIEZO1 agonist Yoda1 (Selleck, Houston, TX, Cat. #S6678).

Techniques: Gene Expression, Knockdown, Quantitative RT-PCR

Journal: JOR Spine

Article Title: Mechanosensitive Ion Channel PIEZO1 Suppresses BMP2 ‐Induced Ossification of the Annulus Fibrosus Cells

doi: 10.1002/jsp2.70168

Figure Lengend Snippet: Activation of Piezo1 by Yoda1 suppresses osteogenic markers in rat AF cells. (A) RT‐qPCR showed downregulation of Runx2 , Osx , and Alp after treatment with 10 μM Yoda1 for 12 h. (B) Western blotting analysis revealed that RUNX2 protein levels decreased in rat AF cells after 24‐h Yoda1 treatment. Figure shows the full‐length blot images. Statistical significance was set at P < 0.05, with significance levels denoted as follows: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: We measured intracellular calcium responses using the calcium indicator dye Fluo‐8 NW (AAT Bioquest, Pleasanton, CA, Cat. #36315) and PIEZO1 agonist Yoda1 (Selleck, Houston, TX, Cat. #S6678).

Techniques: Activation Assay, Quantitative RT-PCR, Western Blot

Journal: JOR Spine

Article Title: Mechanosensitive Ion Channel PIEZO1 Suppresses BMP2 ‐Induced Ossification of the Annulus Fibrosus Cells

doi: 10.1002/jsp2.70168

Figure Lengend Snippet: Activation of Piezo1 by Yoda1 suppresses osteogenic markers in human AF cells. Human AF cells were isolated from nondegenerated (Pfirrmann grade 1) and severely degenerated (Pfirrmann grade 5) intervertebral discs. (A) PIEZO1 mRNA expression showed no significant difference between grade 1 and grade 5 cells. (B–E) Consistent with observations in rat AF cells, treatment with Yoda1 (10 μM, 12 h) significantly downregulated osteogenesis‐related genes ( RUNX2 , OSX ) at the mRNA level and reduced RUNX2 protein expression. Full‐length Western blot images are shown in Figure . Statistical significance was set at P < 0.05, with significance levels denoted as follows: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: We measured intracellular calcium responses using the calcium indicator dye Fluo‐8 NW (AAT Bioquest, Pleasanton, CA, Cat. #36315) and PIEZO1 agonist Yoda1 (Selleck, Houston, TX, Cat. #S6678).

Techniques: Activation Assay, Isolation, Expressing, Western Blot

Journal: JOR Spine

Article Title: Mechanosensitive Ion Channel PIEZO1 Suppresses BMP2 ‐Induced Ossification of the Annulus Fibrosus Cells

doi: 10.1002/jsp2.70168

Figure Lengend Snippet: Transcriptomic analysis of AF cells following Piezo1 activation by Yoda1. (A) PCA showed a clear separation between the control (DMSO) and Yoda1‐treated groups. (B) Volcano plot of DEGs shows 3281 upregulated and 3362 downregulated genes. (C) GSEA reveals enrichment of ossification‐ and calcium signaling‐related pathways, including regulation of ossification, regulation of bone mineralization, and calcineurin‐mediated signaling, in Yoda1‐treated AF cells.

Article Snippet: We measured intracellular calcium responses using the calcium indicator dye Fluo‐8 NW (AAT Bioquest, Pleasanton, CA, Cat. #36315) and PIEZO1 agonist Yoda1 (Selleck, Houston, TX, Cat. #S6678).

Techniques: Activation Assay, Control

Journal: JOR Spine

Article Title: Mechanosensitive Ion Channel PIEZO1 Suppresses BMP2 ‐Induced Ossification of the Annulus Fibrosus Cells

doi: 10.1002/jsp2.70168

Figure Lengend Snippet: Piezo1 activation inhibits BMP2‐induced osteogenesis through calcineurin signaling. (A) Schematic of the experimental schedule for the BMP2 and Yoda1 treatment. (B) RT‐qPCR showed that Yoda1 cotreatment suppressed the BMP2‐induced upregulation of Osx and Alp in AF cells. (C, D) Alizarin Red staining revealed that BMP2 alone enhanced calcium deposition, whereas cotreatment with Yoda1 reduced this staining intensity. (E) Calcineurin phosphatase assay showing that Yoda1 significantly increased calcineurin enzymatic activity compared with DMSO control; this effect was abolished by co‐treatment with cyclosporin A (CsA). (F, G) Immunocytochemistry for p‐Smad1/5/9 showed increased nuclear translocation after BMP2 treatment but significantly decreased BMP2 + Yoda1 cotreated group. Furthermore, cotreatment with the calcineurin inhibitor CsA rescues the nuclear translocation of p‐Smad1/5/9. Statistical significance was set at P < 0.05, with significance levels denoted as follows: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: We measured intracellular calcium responses using the calcium indicator dye Fluo‐8 NW (AAT Bioquest, Pleasanton, CA, Cat. #36315) and PIEZO1 agonist Yoda1 (Selleck, Houston, TX, Cat. #S6678).

Techniques: Activation Assay, Quantitative RT-PCR, Staining, Phosphatase Assay, Activity Assay, Control, Immunocytochemistry, Translocation Assay

Journal: JOR Spine

Article Title: Mechanosensitive Ion Channel PIEZO1 Suppresses BMP2 ‐Induced Ossification of the Annulus Fibrosus Cells

doi: 10.1002/jsp2.70168

Figure Lengend Snippet: Proposed mechanism schema. Piezo1‐mediated calcium influx activates calcineurin, which dephosphorylates p‐Smad1/5/9, inhibiting its nuclear translocation and BMP2 signaling.

Article Snippet: We measured intracellular calcium responses using the calcium indicator dye Fluo‐8 NW (AAT Bioquest, Pleasanton, CA, Cat. #36315) and PIEZO1 agonist Yoda1 (Selleck, Houston, TX, Cat. #S6678).

Techniques: Translocation Assay

Journal: ACS Omega

Article Title: Suppression of Esophageal Squamous Cell Carcinoma Development by Mechanosensitive Protein Piezo1 Downregulation

doi: 10.1021/acsomega.1c00505

Figure Lengend Snippet: Piezo1 expression in tissues. (a) Immunohistochemical images of Piezo1 expression in paracancerous tissues (above) and ESCC tissues (below). (b) Relative expression level of Piezo1 in the cancer genome atlas (TCGA) samples.

Article Snippet: Then, 1 mL of Piezo1 agonist Yoda1 (26.6 μM, Glpbio, USA) was added, and cells were incubated for 10 min.

Techniques: Expressing, Immunohistochemical staining

Journal: ACS Omega

Article Title: Suppression of Esophageal Squamous Cell Carcinoma Development by Mechanosensitive Protein Piezo1 Downregulation

doi: 10.1021/acsomega.1c00505

Figure Lengend Snippet: Construction of Piezo1-downregulated cell lines. (a) Piezo1 mRNA expression in different ESCC cell lines. (b) Protein expression of Piezo1 in transfected EC9706 and EC109 cell lines. (c and d) Relative mRNA expression of Piezo1 in transfected EC9706 and EC109 cell lines. * p < 0.05 vs control and *** p < 0.001 vs control.

Article Snippet: Then, 1 mL of Piezo1 agonist Yoda1 (26.6 μM, Glpbio, USA) was added, and cells were incubated for 10 min.

Techniques: Expressing, Transfection, Control

Journal: ACS Omega

Article Title: Suppression of Esophageal Squamous Cell Carcinoma Development by Mechanosensitive Protein Piezo1 Downregulation

doi: 10.1021/acsomega.1c00505

Figure Lengend Snippet: FI of intracellular Ca 2+ and intracellular Ca 2+ levels increased by Yoda1. (a) FI before in EC109 shRNA-piezo1 and EC109 shRNA-control cells. ** p < 0.001 vs control. (b) FI before in EC9706 shRNA-piezo1 and EC9706 shRNA-control cells. *** p < 0.001 vs control. (c) ΔFI in EC109 shRNA-piezo1 and EC109 shRNA-control cells. *** p < 0.05 vs control. (d) ΔFI in EC9706 shRNA-piezo1 and EC9706 shRNA-control cells. * p < 0.05 vs control.

Article Snippet: Then, 1 mL of Piezo1 agonist Yoda1 (26.6 μM, Glpbio, USA) was added, and cells were incubated for 10 min.

Techniques: shRNA, Control

Journal: ACS Omega

Article Title: Suppression of Esophageal Squamous Cell Carcinoma Development by Mechanosensitive Protein Piezo1 Downregulation

doi: 10.1021/acsomega.1c00505

Figure Lengend Snippet: Migration and invasion of transfected cells. (a) Migration of EC109 shRNA-piezo1 and EC109 shRNA-control cells. (b) Quantitative analysis of panel A, *** p < 0.001 vs control. (c) Invasion of EC109 shRNA-piezo1 and EC109 shRNA-control cells, ** p < 0.01 vs control. (d and e) Invasion and migration of EC9706 shRNA-piezo1 and EC9706 shRNA-control cells, ** p < 0.01 vs control. (f) Protein expression of E-cadherin and N-cadherin in transfected EC109 and EC9706 cells.

Article Snippet: Then, 1 mL of Piezo1 agonist Yoda1 (26.6 μM, Glpbio, USA) was added, and cells were incubated for 10 min.

Techniques: Migration, Transfection, shRNA, Control, Expressing

Journal: ACS Omega

Article Title: Suppression of Esophageal Squamous Cell Carcinoma Development by Mechanosensitive Protein Piezo1 Downregulation

doi: 10.1021/acsomega.1c00505

Figure Lengend Snippet: Piezo1 downregulation affects ESCC cell proliferation, cell cycle progression, and apoptosis. Proliferation was suppressed by Piezo1 downregulation in (a) EC109 cells and (b) EC9706 cells, *** p < 0.001 vs control. (c) Cell cycle distribution of transfected EC109 cells. (d) Analysis of cell cycle distribution in panel C, * p < 0.05 vs control. (e) Cell cycle distribution of transfected EC109 cells. (f) Analysis of cell cycle distribution in panel E, * p < 0.05 vs control. Apoptosis of transfected (g) EC109 cells and (h) EC109 cells. (i) Protein expressions of cleaved-caspase3 and caspase3 in transfected EC109 and EC9706 cells, respectively.

Article Snippet: Then, 1 mL of Piezo1 agonist Yoda1 (26.6 μM, Glpbio, USA) was added, and cells were incubated for 10 min.

Techniques: Control, Transfection

Journal: ACS Omega

Article Title: Suppression of Esophageal Squamous Cell Carcinoma Development by Mechanosensitive Protein Piezo1 Downregulation

doi: 10.1021/acsomega.1c00505

Figure Lengend Snippet: Interaction between p53 and Piezo1, and the protein expression of p53 and Bax. (a) Co-IP between Piezo1 and p53 in EC109 and EC9706 cells. Cell lysates underwent IP using control IgG or the indicated antibody, and the precipitated protein was detected using immunoblotting analysis with the indicated antibody. Cell extracts were used as a positive control (input). (b) Relative mRNA levels of p53 and Bax in transfected EC109 and EC9706 cells, respectively. ** p < 0.01 vs control and * p < 0.05 vs control. (c) Protein expressions of p53 and Bax in transfected EC109 and EC9706 cells, respectively. (d) Relative mRNA expression levels of Piezo1 and p53 in EC109 and (e) EC9706 cell lines after the addition of the p53 inhibitor.

Article Snippet: Then, 1 mL of Piezo1 agonist Yoda1 (26.6 μM, Glpbio, USA) was added, and cells were incubated for 10 min.

Techniques: Expressing, Co-Immunoprecipitation Assay, Control, Western Blot, Positive Control, Transfection

Journal: ACS Omega

Article Title: Suppression of Esophageal Squamous Cell Carcinoma Development by Mechanosensitive Protein Piezo1 Downregulation

doi: 10.1021/acsomega.1c00505

Figure Lengend Snippet: Tumor growth in tumor-bearing mice. (a) Images of tumors harvested from mice shRNA-piezo1 and mice shRNA-control . (b) Final weights of tumors in mice shRNA-piezo and mice shRNA-control . ** p < 0.01 vs control. (c) Tumor volumes measured throughout the experiment. * p < 0.05 vs control. (d) Protein expression of Bax, cleaved-caspase3, caspase3, p53, E-cadherin, and N-cadherin in tumor tissues.

Article Snippet: Then, 1 mL of Piezo1 agonist Yoda1 (26.6 μM, Glpbio, USA) was added, and cells were incubated for 10 min.

Techniques: shRNA, Control, Expressing

Journal: ACS Omega

Article Title: Suppression of Esophageal Squamous Cell Carcinoma Development by Mechanosensitive Protein Piezo1 Downregulation

doi: 10.1021/acsomega.1c00505

Figure Lengend Snippet: Schema of the Piezo1-p53-Bax-Caspase 3 axis.

Article Snippet: Then, 1 mL of Piezo1 agonist Yoda1 (26.6 μM, Glpbio, USA) was added, and cells were incubated for 10 min.

Techniques:

Journal: ACS Omega

Article Title: Suppression of Esophageal Squamous Cell Carcinoma Development by Mechanosensitive Protein Piezo1 Downregulation

doi: 10.1021/acsomega.1c00505

Figure Lengend Snippet: Piezo1 Expression in Cancerous and Paracancerous Tissues

Article Snippet: Then, 1 mL of Piezo1 agonist Yoda1 (26.6 μM, Glpbio, USA) was added, and cells were incubated for 10 min.

Techniques: Expressing

Journal: International Dental Journal

Article Title: Piezo1 Promotes Odontogenic Differentiation of Dental Pulp Stem Cells Under Stress Conditions

doi: 10.1016/j.identj.2025.01.018

Figure Lengend Snippet: Piezo1 mediates the mechanical transduction of DPSCs under single-cell mechanical stimulation. (A) Schematic diagram of the single-cell mechanical stimulation model; (B) microscopic mapping under single-cell mechanical stimulation; (C) DPSCs rapidly deactivated inward current when stimulated with a stimulus (0-8 μm) under single-cell mechanical stimulation; (D and E) with Yoda1 treatment, the peak inward current produced by single-cell mechanical stimulation increased by approximately 150% and was suppressed by approximately 80% after GsMTx4 treatment. (F) With Yoda1 treatment, the current inactivation rate was lower than that in the controls. * P < .05 compared with the control group.

Article Snippet: Yoda1 (a Piezo1-specific agonist, 10 μM; APExBIO), Yoda1 with the Piezo1 blocker GsMTx4 (5 μM; APExBIO), and Yoda1 with the Ca 2+ -chelating agent BAPTA (10 μM; APExBIO) were subsequently used to stimulate the cells.

Techniques: Transduction, Produced, Control

Journal: International Dental Journal

Article Title: Piezo1 Promotes Odontogenic Differentiation of Dental Pulp Stem Cells Under Stress Conditions

doi: 10.1016/j.identj.2025.01.018

Figure Lengend Snippet: Piezo1 regulates the Ca 2+ /CaN/YAP pathway to promote odontogenic differentiation of DPSCs. (A) The colocalization and nucleoplasm shuttle effects of Yoda1-mediated NFATc1 and YAP were detected by cellular immunofluorescence. (B and C) The expression of NFATc1 and YAP in the nucleus of DPSCs was detected by Western blot analysis. (D and E) Alizarin red staining showed that treatment with BAPTA (a Ca 2+ -chelating agent), CsA (a CaN blocker), or Verteporfin (a YAP blocker) reversed the effect of HP on the odontogenic differentiation of DPSCs. (F) Direct binding of the NFATc1 and YAP proteins in DPSCs was confirmed by a protein coimmunoprecipitation (Co-IP) assay. (G and H) Western blot analysis results showing that BAPTA, CsA, and Verteporfin significantly inhibited the expression of CaN, NFAT, YAP, and osteoblast-related proteins (DSPP, DMP1) under HP stimulation, while Verteporfin inhibited the expression of only DSPP, DMP1, and YAP signalling molecules. * P < .05 compared with the NC group.

Article Snippet: Yoda1 (a Piezo1-specific agonist, 10 μM; APExBIO), Yoda1 with the Piezo1 blocker GsMTx4 (5 μM; APExBIO), and Yoda1 with the Ca 2+ -chelating agent BAPTA (10 μM; APExBIO) were subsequently used to stimulate the cells.

Techniques: Immunofluorescence, Expressing, Western Blot, Staining, Binding Assay, Co-Immunoprecipitation Assay

Journal: Scientific Reports

Article Title: The local mechanosensitive response of primary cardiac fibroblasts is influenced by the microenvironment mechanics

doi: 10.1038/s41598-024-60685-4

Figure Lengend Snippet: Morphological and mechanical investigations of cells together with Piezo1 expression for 24 or 48 h. Brightfield images (scale bar: 100 μm) for 24 h ( a ) and for 48 h of cultured cells ( b ) and surface area quantification (N = 61 for 24 h and N = 54 for 48 h) ( c ) show an increment in cell spreading with the time. ( d ) Fluorescence images for 24- and 48-h cultured cells were obtained by staining cells for actin (red), α-SMA (green), and DAPI (blue). At 48 h an increment in α-SMA incorporation into actin stress fibers is observed, as evidenced by the merged image. Scale bar: 40μm. ( e ) Piezo1 expression normalized to GAPDH for 24- and 48-h cultured cells (n = 3). ( f ) Distribution of ΔF/F0 values for all responding cells chemically stimulated with Yoda1 20μM (N = 49 for 24h and 60 for 48h). ( g ) Young’s Modulus measurement for 24- and 48-h cultured cells, N = 87. ( e – g ) demonstrate that neither changes in channel expression nor cellular mechanics are the underlying causes of the different gating behavior between 24 and 48 h.

Article Snippet: For Yoda1 experiment a solution of Yoda1 (CS-5095-5 mg, Chemscene, Monmouth Junction, NJ, USA) in NPSS was injected into the sample at a final concentration of 20 μM.

Techniques: Expressing, Cell Culture, Fluorescence, Staining

Journal: Scientific Reports

Article Title: The local mechanosensitive response of primary cardiac fibroblasts is influenced by the microenvironment mechanics

doi: 10.1038/s41598-024-60685-4

Figure Lengend Snippet: Inhibiting myofibroblasts content within the population lowered the cell elasticity and the channel expression, and, thus, also the mechanical and chemical channel activation. ( a ) α-SMA expression normalized to GAPDH for cells cultured on plastic (CTR) and elastomeric substrates (PDMS), n = 3. ( b ) Piezo1 expression normalized to GAPDH for cells cultured on plastic (CTR) and elastomeric substrates (PDMS), n = 3. ( c ) Young’s Modulus for cells cultured on plastic (CTR) and elastomeric surfaces (PDMS) (N = 17). ( d ) Distribution of ΔF/F 0 values for all responding cells chemically stimulated with Yoda1 20μM (N = 93 for CTR and 76 for PDMS).

Article Snippet: For Yoda1 experiment a solution of Yoda1 (CS-5095-5 mg, Chemscene, Monmouth Junction, NJ, USA) in NPSS was injected into the sample at a final concentration of 20 μM.

Techniques: Expressing, Activation Assay, Cell Culture

Journal: ACS Applied Materials & Interfaces

Article Title: Yoda1-Loaded Microfibrous Scaffolds Accelerate Osteogenesis through Piezo1-F-Actin Pathway-Mediated YAP Nuclear Localization and Functionalization

doi: 10.1021/acsami.5c03093

Figure Lengend Snippet: Schematic illustration of the fabrication process for Yoda1-loaded PCL microfibrous scaffolds and their impact on enhancing in vitro osteogenic differentiation and in vivo osteogenesis.

Article Snippet: This substantially increased the drug presence time compared to the nanocarrier delivery of Yoda1, which was only able to maintain an effective time of 12h, as fabricated by Guan et al.

Techniques: In Vitro, In Vivo

Journal: ACS Applied Materials & Interfaces

Article Title: Yoda1-Loaded Microfibrous Scaffolds Accelerate Osteogenesis through Piezo1-F-Actin Pathway-Mediated YAP Nuclear Localization and Functionalization

doi: 10.1021/acsami.5c03093

Figure Lengend Snippet: Characterization of EHD-printed microfibrous scaffolds. (A) SEM images of the P, PP, and PPY groups. (B) EDS elemental maps of carbon (C), oxygen (O), nitrogen (N), chlorine (Cl), and sulfur (S) in the P, PP, and PPY groups. (C) Quantitative surface morphology and (D) roughness analysis of the P, PP, and PPY groups. (E) XRD analysis of the P, PP, and PPY groups. (F) Tensile stress–strain curves of the P, PP, and PPY groups. (G) Proliferation of MC3T3-E1 cells under different concentrations of Yoda1. (H) Release curves of Yoda1 from the PPY groups. n = 3. ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: This substantially increased the drug presence time compared to the nanocarrier delivery of Yoda1, which was only able to maintain an effective time of 12h, as fabricated by Guan et al.

Techniques:

Journal: ACS Applied Materials & Interfaces

Article Title: Yoda1-Loaded Microfibrous Scaffolds Accelerate Osteogenesis through Piezo1-F-Actin Pathway-Mediated YAP Nuclear Localization and Functionalization

doi: 10.1021/acsami.5c03093

Figure Lengend Snippet: Yoda1-loaded scaffolds mediate YAP nuclear localization through the Piezo1-F-actin pathway. (A) Immunofluorescence staining and (B) semiquantitative analysis of Piezo1 in the P, PP, and PPY groups. (C) Piezo1 gene expression level in the P, PP, and PPY groups. (D) Immunofluorescence staining and (E) semiquantitative analysis of Ca 2+ in the P, PP, and PPY groups. (F) Semiquantitative analysis of F-actin in the P, PP, and PPY groups. (G) Immunofluorescence staining, SEM images of F-actin in the P, PP, and PPY groups. (H) Immunofluorescence staining and (I) semiquantitative analysis of YAP in the P, PP, and PPY groups. (J) Schematic diagram of Cyto-D depolymerization of F-actin. (K) Immunofluorescence staining and (L) semiquantitative analysis of YAP in the PPY group treated with Cyto-D or DMSO. n = 3. ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: This substantially increased the drug presence time compared to the nanocarrier delivery of Yoda1, which was only able to maintain an effective time of 12h, as fabricated by Guan et al.

Techniques: Immunofluorescence, Staining, Gene Expression

Journal: ACS Applied Materials & Interfaces

Article Title: Yoda1-Loaded Microfibrous Scaffolds Accelerate Osteogenesis through Piezo1-F-Actin Pathway-Mediated YAP Nuclear Localization and Functionalization

doi: 10.1021/acsami.5c03093

Figure Lengend Snippet: Chromatin remodeling and lysine acetylation mediated by Piezo1 were necessary for the exercise of YAP functions. (A) Immunofluorescence staining of LaminA/C and (B) semiquantitative analysis in the P, PP, and PPY groups. (C) Nuclear elongation quantitative analysis. (D) Heatmap showing chromatin organization in the nucleus of MC3T3-E1 cells in the P, PP, and PPY groups. (E) Immunofluorescence staining and (F) semiquantitative analysis of acetylated histone lysine in the P, PP, and PPY groups. (G) Schematic diagram of C646 inhibition of lysine acetylation. (H) YAP immunofluorescence staining and (I) semiquantitative analysis of the PPY group treated with C646 or DMSO. (J) Immunofluorescence staining and (K) semiquantitative analysis of COL-I in the PPY group treated with C646 or DMSO. (L) Gene expression level of the COL-I in the PPY group treated with C646 or DMSO. (M) Schematic diagram of Yoda1 activating Piezo1 to promote COL-I expression. n = 3. *** P < 0.001 and **** P < 0.0001.

Article Snippet: This substantially increased the drug presence time compared to the nanocarrier delivery of Yoda1, which was only able to maintain an effective time of 12h, as fabricated by Guan et al.

Techniques: Immunofluorescence, Staining, Inhibition, Gene Expression, Expressing

Journal: ACS Applied Materials & Interfaces

Article Title: Yoda1-Loaded Microfibrous Scaffolds Accelerate Osteogenesis through Piezo1-F-Actin Pathway-Mediated YAP Nuclear Localization and Functionalization

doi: 10.1021/acsami.5c03093

Figure Lengend Snippet: Bone regeneration of Yoda1-loaded scaffolds in vivo . (A) 3D-reconstructed images and sagittal positional maps in the control, P, PP, and PPY groups at 4 and 12 weeks. (B–E) Quantitative analysis of BV, BV/TV, BMD, and Tb.Th in the control, P, PP, and PPY groups at 4 and 12 weeks. (F) HE staining images in the control, P, PP, and PPY groups at 4 and 12 weeks. (G) Masson staining in the control, P, PP, and PPY groups at 4 and 12 weeks. (H, I) Immunochemical staining images in the control, P, PP, and PPY groups at 4 and 12 weeks. (J, K) Percentage of COL-I positive areas in the control, P, PP, and PPY groups at 4 and 12 weeks. (L, M) Percentage of the OCN positive areas in the control, P, PP, and PPY groups at 4 and 12 weeks. n = 6. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: This substantially increased the drug presence time compared to the nanocarrier delivery of Yoda1, which was only able to maintain an effective time of 12h, as fabricated by Guan et al.

Techniques: In Vivo, Control, Staining

Journal: bioRxiv

Article Title: 3D-MINFLUX nanoscopy reveals distinct allosteric mechanisms for activation and modulation of PIEZO1 by Yoda1

doi: 10.1101/2025.07.10.664100

Figure Lengend Snippet: (A) Cartoon depicting the proposed mechanism of action of Yoda1 on PIEZO1 (left) and close-up view of the THU8–9 interface with previously proposed residues lining putative Yoda1 binding sites highlighted in sphere representation. (B) , time course of Ca 2+ -influx (F/F0) evoked by 10 and 100 µM Yoda1 in cells expressing PIEZO1 (left) and P1-A2094W (center) assessed by GCamp8 imaging together with comparison of maximum responses (right). (C) Modulation of PIEZO1 (black, top) and P1.A2094W (yellow, bottom) stretch-evoked currents by 30µM Yoda1. Example traces evoked by incrementing pressure stimuli (left), comparison of peak/sustained ratio (middle) using Mann-Whitney test (PIEZO1: CTL = 4.02, N=14 vs Yoda1 = 1.38, N=15, P=0.0000131) and Student’s t-test(P1.A2094W: CTL = 8.35, N=10 vs Yoda1 = 1.19, N=10, P=0.0033), pressure-response curves (i.e. peak current amplitude at indicated pressure normalized to maximal response, bottom right) and comparison of P 50 values in the absence and presence of Yoda1 using Student’s t-test (PIEZO1: CTL = 26.1 ± 8.9 mmHg, N=14 vs Yoda1 = -16.9 ± 6.65 mmHg, N=13, P=0.006) and Mann-Whitney test (P1.A2094W: CTL = -43.7 ± 10.04 mmHg, N=10 vs Yoda1 = -27.4 ± 6.7 mmHg, N=10, P=0.0005). (D) Modulation of PIEZO1 (black, top) and P1.A2094W (yellow, bottom) poking-evoked currents in whole-cell recordings by 30µM Yoda1. Example traces evoked by incrementing (Δ 800nm, left), comparison of inactivation time constants obtained with exponential decay fit (middle) using Mann-Whitney test (PIEZO1: CTL = 15.9 ± 4.3 ms, N=14 vs Yoda1 = 41.1 ± 21.6, N=12, P=0.000258) and Student’s t-test (P1.A2094W: CTL = 6.39 ± 2.8 ms, N=18 vs Yoda1 = 8.3 ± 2.7, N=20, P=0.0405), displacement-response curves (i.e. peak current amplitude vs. indicated stimulus magnitude; bottom, right) and comparison of mechanical activation thresholds using Mann-Whitney test (PIEZO1: CTL = 4.1 ± 1.2 µm, N=14 vs Yoda1 = 2.8 ± 1.4 µm, N=12, P=0.029; P1.A2094W: CTL = 4.8 ± 1.04 µm, N=18 vs Yoda1 = 3.8 ± 1.1 µm, N=20, P=0.0069).

Article Snippet: To independently test if Yoda1 could potentially bind to these cavities, we next calculated possible docking poses of Yoda1 using the AutoDock Vina , .

Techniques: Binding Assay, Expressing, Imaging, Comparison, MANN-WHITNEY, Activation Assay

Journal: bioRxiv

Article Title: 3D-MINFLUX nanoscopy reveals distinct allosteric mechanisms for activation and modulation of PIEZO1 by Yoda1

doi: 10.1101/2025.07.10.664100

Figure Lengend Snippet: (A) Side view of curved (PDB:7WTL, top left) and flattened (PDB:7WTU, bottom left) PIEZO1 cryo-EM structure with close-up side (middle) and top (right) view of THU8–9 interfaces with the putative binding pockets detected by DoGSite3 algorithm shown in isomesh representation. Amino acid side chains that line the pockets are shown in stick representation. (B) , Close-up views of Yoda1 binding poses detected by AutoDock Vina in the curved (top) and flattened (bottom) PIEZO1 conformation. Note, only the binding poses with the highest scores that align with the binding pockets detected in (A) are shown. Residues that are within a distance of 3.5 Å of Yoda1 are shown in stick representation. Note, F1715 appears to be involved in Yoda1 binding in pocket-2 and pocket-3 in the flat conformation. (C) , AutoDock Vina detects multiple possible docking poses for Yoda1. The bar graph shows the percentage of docking poses in which the indicated amino acids are located within 3.5 Å of Yoda1. Note, F1715 is in close proximity of Yoda1 in 78% of all possible binding poses indicating an important role of F1715 in Yoda1 binding in the flat state.

Article Snippet: To independently test if Yoda1 could potentially bind to these cavities, we next calculated possible docking poses of Yoda1 using the AutoDock Vina , .

Techniques: Cryo-EM Sample Prep, Binding Assay

Journal: bioRxiv

Article Title: 3D-MINFLUX nanoscopy reveals distinct allosteric mechanisms for activation and modulation of PIEZO1 by Yoda1

doi: 10.1101/2025.07.10.664100

Figure Lengend Snippet: (A) Cartoon depicting the overall strategy to resolve Yoda1 induced conformational changes (flattening) measured through changes in interblade distance. Insets illustrate the labelling of PIEZO1 with ALFA tag inserted after H86, and the DNA-PAINT method (left). Individual bound fluorophore is located with high precision in 3 dimensions via 3D-MINFLUX and its iterative process (middle), leading to multiple localisations of the same molecule. (B) Confocal image of a PIEZO1-ALFA-mGL expressing N2a-P1KO cell (left) and corresponding 3D-MINFLUX localizations (right), colored by Z range. Inset shows a triple-labelled PIEZO1, with the 3D scatter plots of the raw localizations and a superimposed cryo-EM structure. The 2D in-plane projections of the 3D data were fitted with a bivariate Gaussian distribution, with their probability densities, enabling determination of the average interblade distance (right). (C) Time-course of the average ± s.e.m. (from N=3-4 independent experiments) of the maximal Yoda1 (50µM) effect on the normalized fluorescence (F/F0) for PIEZO1 (top), A2094W (middle) and V1714A_F1715A (bottom). (D-F) In-plane projections of representative trimers examples of PIEZO1 ( D ), A2094W ( E ) and V1714A_1715A ( F ) from cells treated with cytochalasin-D (CTL) or with cytochalasin-D and Yoda1 (50µM) (left). Comparison of the mean ± s.e.m. interblade distance of the identified trimers after addition of Yoda1 for PIEZO1 ( D , N=93 and 110), A2094W ( E , N=61 and 64) and V1714A_1715A ( F , N=87 and 59), with unpaired t-test (right).

Article Snippet: To independently test if Yoda1 could potentially bind to these cavities, we next calculated possible docking poses of Yoda1 using the AutoDock Vina , .

Techniques: Expressing, Cryo-EM Sample Prep, Fluorescence, Comparison