Journal: Bioactive Materials

Article Title: Mechanically sensitized hydrogel microspheres trigger membrane receptor switch for cartilage repair

doi: 10.1016/j.bioactmat.2026.03.017

Figure Lengend Snippet: OBNC microspheres activate integrin receptors and mechanosensitive calcium channels. A) Mechanistic diagram of integrin activation verified using fluorophores. B-C) Fluorescence microscopy images of MSCs loaded on HAMA or OBNC hydrogel. D) Fluorescence intensity of single cell in each group was quantified. E) Fluorescence microscopy of MSCs loaded on OBNC hydrogel after different treatments. F) Fluorescence intensity in the whole field of view for each group. G) Fluorescence intensity in the single cell for each group. H) Schematic representation of patch clamp experiments. I) Electrical signals generated by MSCs in response to mechanical stimulation. J) Statistical analysis of poking currents (n = 6). K) The concentration of calcium ions in stem cells of different groups as detected by flow cytometry (siRNA1: targeting the TRPM4 gene, siRNA2: targeting the PIEZO1 gene). L) Quantitative analysis of flow cytometric results (∗ symbol represents comparison with HAMA group, # symbol represents comparison with OBNC group). (ns: non-significant, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ## P < 0.01, ### P < 0.001).

Article Snippet: TRPC1 inhibitor (0.3 nM, Pico145, CAS No. 1628287-16-0), TRPM7 inhibitor (1.0 μM, VPC4, CAS No. 945604-76-2), TRPV2 inhibitor (5.0 μM, compound IV2-1, CAS No. 2242724-49-6), TRPM4 inhibitor (1.5 μM, CBA, CAS No. 351424-20-9), PIEZO1 inhibitor (2.5 μM, GsMTx4, CAS No. 1209500-46-8), integrin αvβ5 inhibitor (8.0 nM, Compound 12, CAS No.: 2615912-33-7), integrin αvβ1 inhibitor (0.3 nM, Compound C8, CAS No. 1689540-62-2), integrin α5β1 inhibitor (10 μM, ATN-161, 904763-27-5), and CDK5 inhibitor (5 nM, CDK5-IN-1, 2,639,540-19-3) were purchased from MCE Biotechnology Co., LTD. After the MSCs were treated, the cRGD solution was added at a concentration of 1:200 and incubated in the dark for 15 min, and the results were observed by fluorescence microscopy.

Techniques: Activation Assay, Fluorescence, Microscopy, Single Cell, Patch Clamp, Generated, Concentration Assay, Flow Cytometry, Comparison

Journal: Biomaterials

Article Title: A comparative study assessing neural recording quality and inflammatory tissue response between stiff and flexible microelectrode arrays

doi: 10.1016/j.biomaterials.2025.123929

Figure Lengend Snippet: Heightened piezo1 expression around the stiff implant. A) Representative images showing Piezo1 expression around the flexible (left) and stiff (right) implant site (*). B) Quantification of normalized intensity as a function of distance from the implant (μm). C) Normalized intensity averaged across the first 50 μm from the implant site. 19 images for the flexible and 22 images for the stiff group were analyzed. D) Representative images showing Piezo1 (cyan), Iba-1 (green), GFAP (red), and merged expression around a stiff implant, with the zoomed inset with DAPI (blue) overlay. E) Correlation of total piezo1 expression with total Iba-1 expression per image for flexible (left, blue) and stiff (right, red) groups. The slope is significant for the stiff group, indicating a strong correlation between Piezo1 expression and Iba-1 intensity. F) Correlation of total piezo1 expression with total GFAP expression per image for flexible (left, blue) and stiff (right, red). 4 animals were implanted in each group. C) Two-way ANOVA with Sidak’s multiple comparison test was used, E-F) Data was normalized per image and the mean set to 0. A simple linear regression was performed, dashed lines indicate 95 % confidence intervals, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. The scale bar is 100 μm in A) and D) and 50 μm in the zoomed inset in D). C) Data are mean ± SEM. E-F) Data is sum.

Article Snippet: Antibodies to visualize astrocytes (GFAP, 1:500, Z033401 Dako), macrophage/microglia IBA-1(microglia, 1:500, NC9288364 Fisher), Piezo1 mechanosensitive ion channels (Piezo1,1:100, NBP275617 Novus Biologicals), neuronal nuclei (NeuN, 1:750, CAD69 Abcam), and neural filament (NF200, 1:250 MAB5256 Millipore) were used.

Techniques: Expressing, Comparison

Journal: Biomaterials

Article Title: A comparative study assessing neural recording quality and inflammatory tissue response between stiff and flexible microelectrode arrays

doi: 10.1016/j.biomaterials.2025.123929

Figure Lengend Snippet: Cell-specific intensity analysis. A) Microglia mask of a sample image using Iba-1. Black portions represent the extracted cell structures. This mask was used to identify the sample microglia shown in B). B) Piezo1 (blue) stained image showing activated microglia expressing Piezo1. C) Average within-microglia Piezo1 intensity with distance from the probe center. D) Average within-microglia Iba-1 intensity with distance from the probe center. E) Astrocyte mask of the above image using GFAP. Black portions represent the extracted cell structures. This mask was used to identify the sample astrocyte processes shown in F). F) Piezo1 (blue) stained image showing astrocyte processes expressing Piezo1. G) Average within-astrocyte Piezo1 intensity with distance from the probe center. H) Average within-astrocyte GFAP intensity with distance from the probe center. For all microglia and astrocyte analysis, the increase in intensity with proximity to the injury is highly significant for both probe substrates, and the cells surrounding stiff probes have higher intensity expression than the flexible. I) Piezo1 (red) stained image overlayed with NeuN ROIs produced by Cellpose. J) Piezo1 (red) stained image merged with NeuN (green). K) Average within-neuron Piezo1 intensity with distance from the probe center. Sample DAPI (white) stained image for a L) stiff and M) flexible probe implant. N) Average DAPI circularity index for all images analyzed above. Bar plot is the same data, but binned into two large bins rather than 10 evenly spaced bins. For A) and E), DAPI is overlayed on the mask. DAPI that passes through masked cell structures is seen as red, while DAPI covered by the mask is seen as yellow. Scale bar is 100 μm and 50 μm in zoomed insets in M&N. * Indicates implantation site. For comparisons between stiff and flexible, a two-way ANOVA with Sidak’s multiple comparison test was used. For correlation of intensity with distance from the probe, a Spearman’s r correlation test was used due to the non-normality of many of the datasets, **p < 0.01, ***p < 0.001, ****p < 0.0001. All data is mean ± SEM.

Article Snippet: Antibodies to visualize astrocytes (GFAP, 1:500, Z033401 Dako), macrophage/microglia IBA-1(microglia, 1:500, NC9288364 Fisher), Piezo1 mechanosensitive ion channels (Piezo1,1:100, NBP275617 Novus Biologicals), neuronal nuclei (NeuN, 1:750, CAD69 Abcam), and neural filament (NF200, 1:250 MAB5256 Millipore) were used.

Techniques: Staining, Expressing, Produced, Comparison

Journal: Molecular Medicine Reports

Article Title: Mechanotransducer Piezo1 drives ventilator-induced lung injury in lung epithelial cells via the calcineurin/NFATc3 pathway

doi: 10.3892/mmr.2026.13893

Figure Lengend Snippet: Piezo1 expression is upregulated in the lung epithelium during ventilator-induced lung injury. (A) Representative immunofluorescence images of lung sections from sham-operated control mice and mice subjected to 6 h of mechanical ventilation. Piezo1 protein (red) is constitutively expressed and colocalizes (yellow in merged panels) with the epithelial cell marker CK8 (green). Nuclei were counterstained with DAPI (blue). Scale bar, 100 µm. (B) Representative images of immunohistochemical staining for Piezo1 expression in lung epithelium from sham-operated mice and mice subjected to 6 h of mechanical ventilation. Scale bar, 20 µm (original magnification, ×400). (C) Representative haematoxylin and eosin-stained lung sections from sham-operated and HTV-ventilated mice. Scale bar, 50 µm. (D) Quantitative lung injury score was determined based on histological evaluation of alveolar congestion, haemorrhage, leukocyte infiltration and alveolar wall thickness, each graded from 0 (normal) to 3 (severe). Data are presented as the median (interquartile range); n=6 mice/group and Pvalues were determined by Mann-Whitney U test. Lung injury was further evaluated on the basis of (E) protein concentration, (F) cell number in BALF and (G) wet/dry weight ratio. (H) MPO activity in lung tissue. Proinflammatory cytokines in BALF were evaluated using the following ELISA kits: (I) IL-6, (J) TNF-α and (K) IL-β. (E-K) Data are presented as the mean ± SD, n=6 mice/group. Pvalues were determined by Student's t-test. *P<0.05, **P<0.01, ***P<0.001. BALF, bronchoalveolar lavage fluid; CK8, cytokeratin 8; HTV, high tidal volume; MPO, myeloperoxidase.

Article Snippet: To establish male mice with tamoxifen-inducible, lung epithelial-specific deletion of Piezo1 ( Piezo1 CKO ), Piezo1 F/F mice ( Piezo1 tm2.1Apat/ J; Jackson Laboratory) were bred with Sftpc-Cre ERT mice (Jackson Laboratory) in our laboratory.

Techniques: Expressing, Immunofluorescence, Control, Marker, Immunohistochemical staining, Staining, MANN-WHITNEY, Protein Concentration, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: Molecular Medicine Reports

Article Title: Mechanotransducer Piezo1 drives ventilator-induced lung injury in lung epithelial cells via the calcineurin/NFATc3 pathway

doi: 10.3892/mmr.2026.13893

Figure Lengend Snippet: CS induces Piezo1 expression and epithelial injury markers in lung epithelial cells in vitro . (A) Representative western blot analysis of Piezo1 protein expression in lung epithelial cells under control (unstretched) conditions, and after 3 or 6 h of CS. (B) Semi-quantification of Piezo1 protein expression normalized to that of GAPDH. (C) Piezo1 mRNA expression in sham and stretched cells (after 3 and 6 h). (D) Representative fluorescence images of F-actin (phalloidin, green) and nuclei (DAPI, blue) in control and stretched cells. Scale bar, 20 µm. Proinflammatory cytokine levels of (E) IL-6, (F) TNF-α and (G) IL-1β protein levels in sham versus stretched cells. Data are presented as the mean ± SD, n=6 each. Pvalues were determined by oneway ANOVA. *P<0.05, **P<0.01, ***P<0.001. CS, cyclic stretch.

Article Snippet: To establish male mice with tamoxifen-inducible, lung epithelial-specific deletion of Piezo1 ( Piezo1 CKO ), Piezo1 F/F mice ( Piezo1 tm2.1Apat/ J; Jackson Laboratory) were bred with Sftpc-Cre ERT mice (Jackson Laboratory) in our laboratory.

Techniques: Expressing, In Vitro, Western Blot, Control, Fluorescence

Journal: Molecular Medicine Reports

Article Title: Mechanotransducer Piezo1 drives ventilator-induced lung injury in lung epithelial cells via the calcineurin/NFATc3 pathway

doi: 10.3892/mmr.2026.13893

Figure Lengend Snippet: Lung epithelial-specific Piezo1 deletion attenuates ventilator-induced lung injury. (A) Validation of Piezo1 knockout efficiency in the lung epithelium. Representative immunofluorescence images of lung sections from control and Piezo1 CKO mice showing the expression of the Piezo1 protein (red), the epithelial cell marker CK8 (green), and nuclei (DAPI, blue). Scale bar, 50 µm. (B) Representative haematoxylin and eosin-stained lung sections from control and Piezo1 CKO mice after 6 h of mechanical ventilation. Scale bar, 50 µm. (C) Quantitative histopathological lung injury score. Data are presented as the median (IQR); n=6/group. Data were analysed using the Kruskal-Wallis test followed by Dunn's post hoc test. (D) Total protein concentration in BALF. (E) Total cell counts in the BALF. (F) Lung wet/dry weight ratio. (G) Lung MPO activity. BALF concentrations of the inflammatory cytokines (H) IL-6, (I) TNF-α and (J) IL-1β. (D-J) Data are presented as the mean ± SD, n=6 mice/group. Pvalues were determined by oneway ANOVA. *P<0.05, **P<0.01, ***P<0.001. BALF, bronchoalveolar lavage fluid; CK8, cytokeratin 8; CKO, conditional; HTV, high tidal volume; MPO, myeloperoxidase.

Article Snippet: To establish male mice with tamoxifen-inducible, lung epithelial-specific deletion of Piezo1 ( Piezo1 CKO ), Piezo1 F/F mice ( Piezo1 tm2.1Apat/ J; Jackson Laboratory) were bred with Sftpc-Cre ERT mice (Jackson Laboratory) in our laboratory.

Techniques: Biomarker Discovery, Knock-Out, Immunofluorescence, Control, Expressing, Marker, Staining, Protein Concentration, Activity Assay

Journal: Molecular Medicine Reports

Article Title: Mechanotransducer Piezo1 drives ventilator-induced lung injury in lung epithelial cells via the calcineurin/NFATc3 pathway

doi: 10.3892/mmr.2026.13893

Figure Lengend Snippet: Genetic inhibition of Piezo1 attenuates mechanical stretch-induced cytoskeletal disruption and the inflammatory response in lung epithelial cells in vitro . (A) Representative western blotting and (B) semi-quantitative analysis demonstrating efficient knockdown of Piezo1 protein expression in MLE-12 cells transfected with shPiezo1 compared with that in cells transfected with Scr-shRNA. (C) Piezo1 mRNA expression levels measured by quantitative PCR in cells transfected with shPiezo1 compared with those in cells transfected with Scr-shRNA. Proinflammatory cytokine levels in cell culture supernatants from Scr-shRNA and shPiezo1 cells under control conditions or after 6 h of CS: (D) IL-6, (E) TNF-α and (F) IL-1β protein levels. (G) Representative fluorescence images showing F-actin morphology (phalloidin, green) and nuclei (DAPI, blue) in Scr-shPiezo1 and shPiezo1 cells under control (unstretched) conditions or after 6 h of CS. Scale bar, 20 µm. Data are presented as the mean ± SD, n=6 each. Pvalues were determined by oneway ANOVA. **P<0.01, ***P<0.001. CS, cyclic stretch; Scr, scrambled; sh, short hairpin.

Article Snippet: To establish male mice with tamoxifen-inducible, lung epithelial-specific deletion of Piezo1 ( Piezo1 CKO ), Piezo1 F/F mice ( Piezo1 tm2.1Apat/ J; Jackson Laboratory) were bred with Sftpc-Cre ERT mice (Jackson Laboratory) in our laboratory.

Techniques: Inhibition, Disruption, In Vitro, Western Blot, Knockdown, Expressing, Transfection, shRNA, Real-time Polymerase Chain Reaction, Cell Culture, Control, Fluorescence

Journal: Molecular Medicine Reports

Article Title: Mechanotransducer Piezo1 drives ventilator-induced lung injury in lung epithelial cells via the calcineurin/NFATc3 pathway

doi: 10.3892/mmr.2026.13893

Figure Lengend Snippet: Piezo1 mediates CS-induced Ca 2+ influx in epithelial cells. (A) Quantitative analysis of cytosolic Ca 2+ levels (ΔF/F 0 ) measured using a microplate reader in Fluo-3 AM-loaded MLE-12 cells treated with the Piezo1 agonist Yoda1 (10 µM) for 0, 15 and 30 min. (B) Representative fluorescence microscopy images of Fluo-3 AM-loaded MLE-12 cells at 30 min following the onset of CS Scale bar, 50 µm. (C and D) Both genetic knockdown of Piezo1 (with shPiezo1) and pharmacological inhibition with GsMTx4 (5 µM) attenuated the Ca 2+ elevation induced by 30-min CS. (E) Genetic knockdown of Piezo1 (with shPiezo1) attenuated mechanical stretch-induced calcineurin activation after 6 h. (F-H) Pharmacological inhibition of calcineurin with CsA (10 µM) or FK506 (10 µM) significantly reduced mechanical stretch -induced cytokine production after 6 h. (I) Disruption of the F-actin network induced by 6-h mechanical stretching was reversed by pretreatment with CsA or FK506. Scale bar, 20 µm. Data are presented as the mean ± SD, n=6 each. Pvalues were determined by oneway ANOVA. *P<0.05, ***P<0.001. CS, cyclic stretch; CsA, cyclosporine A; Scr, scrambled; sh, short hairpin.

Article Snippet: To establish male mice with tamoxifen-inducible, lung epithelial-specific deletion of Piezo1 ( Piezo1 CKO ), Piezo1 F/F mice ( Piezo1 tm2.1Apat/ J; Jackson Laboratory) were bred with Sftpc-Cre ERT mice (Jackson Laboratory) in our laboratory.

Techniques: Fluorescence, Microscopy, Knockdown, Inhibition, Activation Assay, Disruption

Journal: Molecular Medicine Reports

Article Title: Mechanotransducer Piezo1 drives ventilator-induced lung injury in lung epithelial cells via the calcineurin/NFATc3 pathway

doi: 10.3892/mmr.2026.13893

Figure Lengend Snippet: Piezo1 mediates CS-induced inflammation via calcineurin/NFATc3 signalling. (A) Immunofluorescence screening of the nuclear translocation of NFAT isoforms (NFATc1-4) upon CS. NFATc3 showed the most prominent nuclear translocation. Scale bar, 10 µm. (B) Representative western blot images showing NFATc3 protein levels in the nuclear and cytoplasmic fractions of MLE-12 cells transfected with Scr-shRNA or shPiezo1, with or without CS (6 h). GAPDH and Lamin B1 were used as loading controls for cytoplasmic and nuclear fractions, respectively. (C) Semi-quantitative analysis of cytoplasmic NFATc3 protein expression normalized to GAPDH. (D) Semi-quantitative analysis of nuclear NFATc3 protein expression normalized to Lamin B1. (E) Representative western blot images showing NFATc3 protein levels in the nuclear and cytoplasmic fractions of MLE-12 cells treated with or without calcineurin inhibitors (CsA, 10 µM; FK506, 10 µM) prior to CS (6 h). GAPDH and Lamin B1 were used as loading controls for cytoplasmic and nuclear fractions, respectively. (F) Semi-quantitative analysis of cytoplasmic NFATc3 protein expression normalized to GAPDH. (G) Semi-quantitative analysis of nuclear NFATc3 protein expression normalized to Lamin B1. Data are presented as the mean ± SD, n=6 each. Pvalues were determined by oneway ANOVA. *P<0.05, **P<0.01, ***P<0.001. CS, cyclic stretch; CsA, cyclosporine A; Scr, scrambled; sh, short hairpin.

Article Snippet: To establish male mice with tamoxifen-inducible, lung epithelial-specific deletion of Piezo1 ( Piezo1 CKO ), Piezo1 F/F mice ( Piezo1 tm2.1Apat/ J; Jackson Laboratory) were bred with Sftpc-Cre ERT mice (Jackson Laboratory) in our laboratory.

Techniques: Immunofluorescence, Translocation Assay, Western Blot, Transfection, shRNA, Expressing

Journal: Bone Research

Article Title: KDM6B safeguards mineralized tissue homeostasis from mechanical stress through epigenetic control of PIEZO1-mediated mechanotransduction in the mouse incisor

doi: 10.1038/s41413-026-00544-2

Figure Lengend Snippet: Loss of Kdm6b leads to apoptosis of TACs via PIEZO1-mediated calcium hyperactivity under mechanical load. a Heatmap of bulk RNA-seq data from the proximal region of control and Gli1-CreER T2 ;Kdm6b fl/fl mice at 5 dpt. b Top 10 upregulated signaling pathways identified by KEGG analysis of differentially expressed genes. c , d Immunostaining of p-CaMKII in control and Gli1-CreER T2 ;Kdm6b fl/fl incisors. c’ , d’ Magnified views of boxed regions. White dotted lines outline the cervical loop. Yellow arrows indicate positive signals. Scale bars, 50 μm. e Western blot and quantification of p-CaMKII and CaMKII in the proximal incisor region (mean ± SEM, n = 3; P = 0.014 0). f Heatmap showing expression of calcium signaling-related genes in control and Gli1-CreER T2 ;Kdm6b fl/fl incisors ( n = 3; individual P -values in Fig. ). g , h Piezo1 in situ hybridization in control and Gli1-CreER T2 ;Kdm6b fl/fl incisors. g’ , h’ Magnified views of boxed regions. White dotted lines outline the cervical loop. Yellow arrows indicate positive signals. Scale bars, 50 μm. i Schematic of time-lapse calcium imaging (Fluo-4) in dental mesenchymal cells. j , k Representative calcium traces from mesenchymal cells of control and Gli1-CreER T2 ;Kdm6b fl/fl mice before and after Yoda1 stimulation. Each trace represents one cell ( n = 15). l Quantification of spontaneous calcium spikes calculated as [(F max -F 0 )/F 0 ] (mean ± SEM, n = 15; P = 0.006 9)

Article Snippet: These gRNAs were similarly cloned into the pCas-Guide-Puro-CRISPRi vector to generate a custom Piezo1 CRISPRi kit (Origene Technologies, CW312380 ).

Techniques: RNA Sequencing, Control, Protein-Protein interactions, Immunostaining, Western Blot, Expressing, In Situ Hybridization, Imaging

Journal: Bone Research

Article Title: KDM6B safeguards mineralized tissue homeostasis from mechanical stress through epigenetic control of PIEZO1-mediated mechanotransduction in the mouse incisor

doi: 10.1038/s41413-026-00544-2

Figure Lengend Snippet: KDM6B regulates PIEZO1 expression and tissue homeostasis through H3K27me3. a , b H3K27me3 immunofluorescence in control and Gli1-CreER T2 ;Kdm6b fl/fl incisors. a’ , b’ magnified views of boxed regions. White dotted lines outline the cervical loop. Asterisk indicates absence of signal. Yellow arrows indicate positive cells. Scale bars, 50 μm. c Western blot analysis of H3K27me3 and H3 in the proximal incisor region. d –f TUNEL staining in control, Kdm6b mutant, and Ezh2 rescue incisors at 1 wpt. d’ –f’ Magnified views of boxed regions. White dotted lines outline the cervical loop. Yellow arrows indicate TUNEL + cells. Scale bars, 50 μm. g Quantification of TUNEL + cells in the dental mesenchymal region (mean ± SEM, n = 6; control vs. Kdm6b mutant, P < 0.000 1; Kdm6b mutant vs. Ezh2 rescue, P < 0.000 1; control vs. Ezh2 rescue, P = 0.019 7). h –j Piezo1 in situ hybridization in the incisors of control, Kdm6b mutant, and Ezh2 rescue mice. h’ –j’ Magnified views of boxed regions. White dotted lines outline the cervical loop. Yellow arrows indicate positive signals. k Relative Piezo1 mRNA expression (mean ± SEM. n = 3; control vs Kdm6b mutant, P = 0.001 5; Kdm6b mutant vs Ezh2 rescue, P = 0.004 9; control vs Ezh2 rescue, P = 0.405 4. Kdm6b mutant: Gli1-CreER T2 ;Kdm6b fl/fl ; Ezh2 rescue: Gli1-CreER T2 ;Kdm6b fl/fl ;Ezh2 fl/+ )

Article Snippet: These gRNAs were similarly cloned into the pCas-Guide-Puro-CRISPRi vector to generate a custom Piezo1 CRISPRi kit (Origene Technologies, CW312380 ).

Techniques: Expressing, Immunofluorescence, Control, Western Blot, TUNEL Assay, Staining, Mutagenesis, In Situ Hybridization

Journal: Bone Research

Article Title: KDM6B safeguards mineralized tissue homeostasis from mechanical stress through epigenetic control of PIEZO1-mediated mechanotransduction in the mouse incisor

doi: 10.1038/s41413-026-00544-2

Figure Lengend Snippet: KDM6B epigenetically regulates BMI1 to silence PIEZO1 expression and maintain tissue homeostasis. a CUT&RUN profiles of H3K27me3 in the proximal incisor region of control and Gli1-CreER T2 ;Kdm6b fl/fl mice at 5 dpt. TSS: Transcription start sites. TES: Transcription end sites. b Venn diagram showing overlap between genes with increased H3K27me3 enrichment in CUT&RUN and downregulated genes in RNA-seq from Gli1-CreER T2 ;Kdm6b fl/fl incisors. c Heatmap of overlapping genes expressed in TACs in control and Gli1-CreER T2 ;Kdm6b fl/fl mice. d Co-staining of Bmi1 (white) and Kdm6b (red) in the wild-type mouse incisors. d’ Magnified view of boxed region. White dotted lines outline the cervical loop. Yellow arrows indicate double-positive cells. Scale bars, 50 μm. e CUT&RUN tracks showing H3K27me3 enrichment at the Bmi1 locus in control and Gli1-CreER T2 ;Kdm6b fl/fl incisors. IgG serves as a negative control. Red lines indicate CRISPRi gRNA target sites. f RT-qPCR analysis of Bmi1 expression following CRISPRi targeting (mean ± SEM, n = 3; gRNA1, P = 0.034 6; gRNA2, P = 0.709 1; gRNA3, P = 0.058 7). g –i Bmi1 in situ hybridization in control, Kdm6b mutant, and Ezh2 rescue incisors. g’ –i’ Magnified views of boxed regions. White dotted lines outline the cervical loop. Yellow arrows indicate positive signals. Asterisk indicates absence of signal. j Relative Bmi1 mRNA expression in the three genotypes (mean ± SEM, n = 3; control vs Kdm6b mutant, P = 0.004 2; Kdm6b mutant vs Ezh2 rescue, P = 0.000 2; control vs Ezh2 rescue, P = 0.012 8). k Piezo1 expression in dental mesenchymal cells after 3 days of control or Bmi1 siRNA treatment (mean ± SEM, n = 3; P = 0.036 4). l Piezo1 expression after vector or Bmi1 plasmid treatment (mean ± SEM, n = 3; ctrl+vector vs. ctrl+ Bmi1 , P = 0.022 6; ctrl+vector vs. mutant+vector, P = 0.000 8; ctrl+vector vs. mutant+ Bmi1 , P = 0.838 4; mutant+vector vs. mutant+ Bmi1 , P = 0.000 9). m CUT&RUN profiles showing BMI1 enrichment at the Piezo1 locus (IgG negative control and two BMI1 replicates). Red lines indicate CRISPRi gRNA target sites. n RT-qPCR analysis of Piezo1 expression following CRISPRi targeting of BMI1-binding regions (mean ± SEM, n = 3; gRNA1, P = 0.012 3 ; gRNA2, P = 0.029 7). Kdm6b mutant: Gli1-CreER T2 ;Kdm6b fl/fl . Ezh2 rescue: Gli1-CreER T2 ;Kdm6b fl/fl ;Ezh2 fl/+

Article Snippet: These gRNAs were similarly cloned into the pCas-Guide-Puro-CRISPRi vector to generate a custom Piezo1 CRISPRi kit (Origene Technologies, CW312380 ).

Techniques: Expressing, Control, RNA Sequencing, Staining, Negative Control, Quantitative RT-PCR, In Situ Hybridization, Mutagenesis, Plasmid Preparation, Binding Assay

Journal: Bone Research

Article Title: KDM6B safeguards mineralized tissue homeostasis from mechanical stress through epigenetic control of PIEZO1-mediated mechanotransduction in the mouse incisor

doi: 10.1038/s41413-026-00544-2

Figure Lengend Snippet: Haploinsufficiency of Piezo1 rescues TAC fate and tissue homeostasis in Gli1-CreER T2 ;Kdm6b fl/fl mice. a –c Micro-CT images of incisors from control, Kdm6b mutant, and Piezo1 rescue mice at 2 mpt. a’ –c’ Coronal sections through the first molar furcation (white lines). Yellow arrows indicate dental pulp. Scale bars, 1 mm. d –f H&E staining of incisors from the three groups. d’ –f’ Magnified views of boxed regions. Scale bars, 50 μm. g –i Dspp (red) in situ hybridization (red) in the incisors from the three groups. White dotted lines show the distance between the cervical loop bending point and the odontoblast initiation. Scale bars: 50 µm. j Quantification of pulp cavity diameter (mean ± SEM, n = 6; control vs. Kdm6b mutant, P < 0.000 1; Kdm6b mutant vs. Piezo1 rescue, P = 0.001 1; control vs. Piezo1 rescue, P = 0.078 6). k Quantification of the distance between Dspp + cells and the cervical loop (mean ± SEM. n = 6; control vs. Kdm6b mutant, P = 0.003 0; Kdm6b mutant vs. Piezo1 rescue, P = 0.004 4; control vs. Piezo1 rescue, P = 0.977 8). l – n TUNEL staining in the incisors from the three groups at 1 wpt. l’ –n’ Magnified views of boxed regions. White dotted lines outline the cervical loop. Yellow arrows indicate TUNEL + cells. Scale bars: 50 µm. o –q Immunostaining of p-CaMKⅡ in the incisors of the three groups at 1 wpt. o’ –q’ Magnified views of boxed regions. White dotted lines outline the cervical loop. Yellow arrows indicate the positive signals. Scale bars: 50 µm. r Western blot of p-CaMKⅡ, CaMKⅡ, and GAPDH in the proximal regions of the incisors from the three groups. s Quantification of TUNEL + cells in dental mesenchymal region (mean ± SEM. n = 6; control vs. Kdm6b mutant, P < 0.000 1; Kdm6b mutant vs. Piezo1 rescue, P < 0.000 1; control vs. Piezo1 rescue, P = 0.019 5). t Quantification of p-CaMKⅡ and CaMKⅡ protein levels (mean ± SEM. n = 3; control vs. Kdm6b mutant, P = 0.036 0; Kdm6b mutant vs. Piezo1 rescue, P = 0.101 2; control vs. Piezo1 rescue, P = 0.699 0). Kdm6b mutant: Gli1-CreER T2 ;Kdm6b fl/fl . Piezo1 rescue: Gli1-CreER T2 ;Kdm6b fl/fl ;Piezo1 fl/+

Article Snippet: These gRNAs were similarly cloned into the pCas-Guide-Puro-CRISPRi vector to generate a custom Piezo1 CRISPRi kit (Origene Technologies, CW312380 ).

Techniques: Micro-CT, Control, Mutagenesis, Staining, In Situ Hybridization, TUNEL Assay, Immunostaining, Western Blot

Journal: Bone Research

Article Title: KDM6B safeguards mineralized tissue homeostasis from mechanical stress through epigenetic control of PIEZO1-mediated mechanotransduction in the mouse incisor

doi: 10.1038/s41413-026-00544-2

Figure Lengend Snippet: Schematic representation of KDM6B safeguarding tissue homeostasis to mechanical stress through epigenetic control of PIEZO1-mediated mechanotransduction. Using the mouse incisor as a model of mechanical loading, we reveal that within TACs, Kdm6b demethylates H3K27me3, thereby relieving the repression of the Bmi1 gene. Normal BMI1 inhibits Piezo1 expression. This maintains physiological PIEZO1 levels, ensuring calibrated Ca 2+ influx for proliferation and differentiation. In contrast, loss of Kdm6b leads to an accumulation of H3K27me3 at the Bmi1 promoter region, which silences Bmi1 expression and diminishes BMI1 formation. This reduction results in pathologically increased PIEZO1 ion channels in the membrane. The subsequent Ca 2+ overload triggers TAC apoptosis while reducing proliferation and differentiation. Ultimately, these molecular events compromise tissue homeostasis. Schematic created with BioRender.com. Ho, T. (2026) https://BioRender.com/8mzv4a3

Article Snippet: These gRNAs were similarly cloned into the pCas-Guide-Puro-CRISPRi vector to generate a custom Piezo1 CRISPRi kit (Origene Technologies, CW312380 ).

Techniques: Control, Expressing, Membrane