piezo1 protein  (Proteintech)

Journal: Materials Today Bio

Article Title: Stiff matrix promotes lung cancer cell migration through down-regulating the Piezo1 channel expression to facilitate Ca 2+ -dependent filopodia formation

doi: 10.1016/j.mtbio.2026.102786

Figure Lengend Snippet: The Piezo1 expression profile in ovary tumor, breast tumor, brain tumor, liver tumor, ovary tumor, lung tumor and matched healthy tissues, based on analysis of GENT2 datasets. Data were presented as the mean ± SD. ∗∗∗P < 0.001.

Article Snippet: Briefly, cells were harvested and fixed with 4 % paraformaldehyde for 30 min, then permeabilized with 0.3 % Triton X-100 for 10 min. After that, cells were incubated with the primary polyclonal antibody recognizing the extracellular domain of Piezo1 protein (1:100 dilution, #15939–1-AP, Proteintech, USA) or p-SSH1 (p Ser978) (1:100 dilution, #NBP3-23411, Novus Biologicals) for 40 min. Then, cells were incubated with FITC-labeled goat anti-rabbit secondary antibody solution (1:100 dilution, #A0562, Beyotime, China) at room temperature for 1 h away from light.

Techniques: Expressing

Journal: Materials Today Bio

Article Title: Stiff matrix promotes lung cancer cell migration through down-regulating the Piezo1 channel expression to facilitate Ca 2+ -dependent filopodia formation

doi: 10.1016/j.mtbio.2026.102786

Figure Lengend Snippet: Stiff substrate promotes A549 and H460 cell migration and down-regulates Piezo1 channel e xpression. (A–D) Transwell assay of the effects of substrate stiffness on cell migration. Representative images of migrated cells stained with crystal violet (10x, A-B) and statistical analysis of data from three independent experiments (C–D). Scale bar: 50 μm. (E–H) Flow cytometry assessing the effects of substrate stiffness on cell surface Piezo1 protein expression. Representative images of flow cytometry (E–F) and statistical analysis of data from three (G–F) independent experiments. All data were normalized to that of 3 kPa group. Data were presented as mean ± SD. ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001; ∗∗∗ P < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Briefly, cells were harvested and fixed with 4 % paraformaldehyde for 30 min, then permeabilized with 0.3 % Triton X-100 for 10 min. After that, cells were incubated with the primary polyclonal antibody recognizing the extracellular domain of Piezo1 protein (1:100 dilution, #15939–1-AP, Proteintech, USA) or p-SSH1 (p Ser978) (1:100 dilution, #NBP3-23411, Novus Biologicals) for 40 min. Then, cells were incubated with FITC-labeled goat anti-rabbit secondary antibody solution (1:100 dilution, #A0562, Beyotime, China) at room temperature for 1 h away from light.

Techniques: Migration, Transwell Assay, Staining, Flow Cytometry, Expressing

Journal: Materials Today Bio

Article Title: Stiff matrix promotes lung cancer cell migration through down-regulating the Piezo1 channel expression to facilitate Ca 2+ -dependent filopodia formation

doi: 10.1016/j.mtbio.2026.102786

Figure Lengend Snippet: Piezo1 channel negatively regulates substrate stiffness-induced A549 cell migration. (A, D) Piezo1 channel blockade with GsMTx4 promotes cell migration on both soft and stiff substrates. (B, E) Piezo1 channel activation with Yoda 1 inhibits cell migration on both soft and stiff substrates. (C, F) Piezo1 channel knockdown with specific siRNA transfection promotes cell migration on both soft and stiff substrates. Representative images of migrated cells stained with crystal violet (10x, A-C) and statistical analysis of data from three independent experiments (D–F). Scale bar: 50 μm. All data were normalized to the 3 kPa group. Data were presented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Briefly, cells were harvested and fixed with 4 % paraformaldehyde for 30 min, then permeabilized with 0.3 % Triton X-100 for 10 min. After that, cells were incubated with the primary polyclonal antibody recognizing the extracellular domain of Piezo1 protein (1:100 dilution, #15939–1-AP, Proteintech, USA) or p-SSH1 (p Ser978) (1:100 dilution, #NBP3-23411, Novus Biologicals) for 40 min. Then, cells were incubated with FITC-labeled goat anti-rabbit secondary antibody solution (1:100 dilution, #A0562, Beyotime, China) at room temperature for 1 h away from light.

Techniques: Migration, Activation Assay, Knockdown, Transfection, Staining

Journal: Materials Today Bio

Article Title: Stiff matrix promotes lung cancer cell migration through down-regulating the Piezo1 channel expression to facilitate Ca 2+ -dependent filopodia formation

doi: 10.1016/j.mtbio.2026.102786

Figure Lengend Snippet: Piezo1 channel negatively regulates stiff substrate-induced filopodia formation in A 549 cells. (A, C, D) Piezo1 channel blockade with GsMTx4 further promotes filopodia formation in cells on both soft and stiff substrates. (B, E, F) Piezo1 channel activation with Yoda 1 further inhibits filopodia formation in cells on stiff substrates but has no effect in cells on soft substrates. Representative images of filopodia morphology (A and B) and statistical analysis of the filopodia length (C and E) and number (D and F) from indicated number of cells. Red, F‐actin staining with rhodamine-labeled phalloidin; blue, nucleus staining with Hoechst 33342. All data were normalized to that of the 3 kPa group. Scale bar: 20 μm. Data were presented as mean ± SD. ∗ P < 0.05; ∗∗∗ P < 0.001; ns, not significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Briefly, cells were harvested and fixed with 4 % paraformaldehyde for 30 min, then permeabilized with 0.3 % Triton X-100 for 10 min. After that, cells were incubated with the primary polyclonal antibody recognizing the extracellular domain of Piezo1 protein (1:100 dilution, #15939–1-AP, Proteintech, USA) or p-SSH1 (p Ser978) (1:100 dilution, #NBP3-23411, Novus Biologicals) for 40 min. Then, cells were incubated with FITC-labeled goat anti-rabbit secondary antibody solution (1:100 dilution, #A0562, Beyotime, China) at room temperature for 1 h away from light.

Techniques: Activation Assay, Staining, Labeling

Journal: Materials Today Bio

Article Title: Stiff matrix promotes lung cancer cell migration through down-regulating the Piezo1 channel expression to facilitate Ca 2+ -dependent filopodia formation

doi: 10.1016/j.mtbio.2026.102786

Figure Lengend Snippet: Piezo1 channel mediates substrate stiffness-induced change in [Ca 2+ ] i in A 549 cells. (A, B) Cells showed the higher and lower [Ca 2+ ] i in cells on soft and stiff substrates, respectively. (C, D) Piezo1 channel blockade with GsMTx4 reduces [Ca 2+ ] i in cells on both soft and stiff substrates. Representative Ca 2+ images (A, C and E) and statistical analysis of [Ca 2+ ] i in indicated numbers of cells (B and D). Scale bar: 50 μm. All data were normalized to that of 3 kPa group. Data were presented as mean ± SD. ∗∗∗ P < 0.001; ns, not significant.

Article Snippet: Briefly, cells were harvested and fixed with 4 % paraformaldehyde for 30 min, then permeabilized with 0.3 % Triton X-100 for 10 min. After that, cells were incubated with the primary polyclonal antibody recognizing the extracellular domain of Piezo1 protein (1:100 dilution, #15939–1-AP, Proteintech, USA) or p-SSH1 (p Ser978) (1:100 dilution, #NBP3-23411, Novus Biologicals) for 40 min. Then, cells were incubated with FITC-labeled goat anti-rabbit secondary antibody solution (1:100 dilution, #A0562, Beyotime, China) at room temperature for 1 h away from light.

Techniques:

Journal: Materials Today Bio

Article Title: Stiff matrix promotes lung cancer cell migration through down-regulating the Piezo1 channel expression to facilitate Ca 2+ -dependent filopodia formation

doi: 10.1016/j.mtbio.2026.102786

Figure Lengend Snippet: Piezo1 channel mediates a strong but weak calcium influx induced by soft and stiff substrates, respectively. (A and B) Extrcellular Ca 2+ influx in cells on soft and stiff substrates. (C and D) Piezo1 blockade with GsMTx4 abolished the difference in Ca 2+ influx between cells on soft and stiff substrates. Representative tracces showing change in [Ca 2+ ] i (A and C) and statistical analysis of the maximal change in [Ca 2+ ] i in indicated numbers of cells (B and D). Cells were cultured in medium with or without 2.5 μM GsMTx4 containment for 48 h. Cells loaded with Fluo4-AM were imaged with 5 s interval in Ca 2+ -free buffer for 1 min and further 4 min upon addition of 2 mM CaCl 2 . Scale bar: 50 μm. All data were normalized to that ones prior to addition of CaCl 2 . Data were presented as mean ± SD. ∗∗∗ P < 0.001.

Article Snippet: Briefly, cells were harvested and fixed with 4 % paraformaldehyde for 30 min, then permeabilized with 0.3 % Triton X-100 for 10 min. After that, cells were incubated with the primary polyclonal antibody recognizing the extracellular domain of Piezo1 protein (1:100 dilution, #15939–1-AP, Proteintech, USA) or p-SSH1 (p Ser978) (1:100 dilution, #NBP3-23411, Novus Biologicals) for 40 min. Then, cells were incubated with FITC-labeled goat anti-rabbit secondary antibody solution (1:100 dilution, #A0562, Beyotime, China) at room temperature for 1 h away from light.

Techniques: Cell Culture

Journal: Materials Today Bio

Article Title: Stiff matrix promotes lung cancer cell migration through down-regulating the Piezo1 channel expression to facilitate Ca 2+ -dependent filopodia formation

doi: 10.1016/j.mtbio.2026.102786

Figure Lengend Snippet: Piezo1 channel regulates stiff substrate-induced phosphorylation of coflilin through reducing the [Ca 2+ ] i in A 549 cells. (A–C) Stiff substrate induces phosphorylation of cofilin (C), without effect on its expression (B). (D–G) Stiff substrate-induced phosphorylation of coflilin is enhanced by Piezo1 channel blockade with GsMTx4 (D and E) but attenuated by Piezo1 channel activation with Yoda-1 (F and G). (H, I) Chelation of intracellular Ca 2+ with BAPTA-AM promotes cofilin phosphorylation in cells on both soft and stiff substrates. Representative images of western blotting (A, D, F and H) and statistical analysis of data from three independent experiments (B, C, E, G and I). All data were normalized to that of the 3 kPa group. Data are presented as mean ± SD. ∗ P < 0.05; ∗ P < 0.01; ∗∗∗ P < 0.001.

Article Snippet: Briefly, cells were harvested and fixed with 4 % paraformaldehyde for 30 min, then permeabilized with 0.3 % Triton X-100 for 10 min. After that, cells were incubated with the primary polyclonal antibody recognizing the extracellular domain of Piezo1 protein (1:100 dilution, #15939–1-AP, Proteintech, USA) or p-SSH1 (p Ser978) (1:100 dilution, #NBP3-23411, Novus Biologicals) for 40 min. Then, cells were incubated with FITC-labeled goat anti-rabbit secondary antibody solution (1:100 dilution, #A0562, Beyotime, China) at room temperature for 1 h away from light.

Techniques: Phospho-proteomics, Expressing, Activation Assay, Western Blot

Journal: Materials Today Bio

Article Title: Stiff matrix promotes lung cancer cell migration through down-regulating the Piezo1 channel expression to facilitate Ca 2+ -dependent filopodia formation

doi: 10.1016/j.mtbio.2026.102786

Figure Lengend Snippet: Piezo1 channel regulates stiff substrate-induced phosphorylation of coflilin through attenuating the Ca 2+ -dependent CaN/SSH activation in A 549 cells. (A–B) CaN inhibition with CsA promotes cofilin phosphorylation in cells on both soft and stiff substrates. (C–D) CaN activity was decreased on stiff substrate, and further decreased on soft and stiff substrates by Piezo1 channel blockade with GsMTx4 (B) or Chelation of intracellular Ca 2+ with BAPTA-AM (D), respectivley. (E–F) The p-SSH1 was incrased on stiff substrates, and further increased on soft and stiff substrates by CaN inhibition with CsA. Representative images of western blotting (A) and flow cytometry (E), and statistical analysis of data from three independent experiments (B, C, D and F). All data were normalized to that of 3 kPa group. Data are presented as mean ± SD. ∗ P < 0.05; ∗ P < 0.01; ∗∗∗ P < 0.001.

Article Snippet: Briefly, cells were harvested and fixed with 4 % paraformaldehyde for 30 min, then permeabilized with 0.3 % Triton X-100 for 10 min. After that, cells were incubated with the primary polyclonal antibody recognizing the extracellular domain of Piezo1 protein (1:100 dilution, #15939–1-AP, Proteintech, USA) or p-SSH1 (p Ser978) (1:100 dilution, #NBP3-23411, Novus Biologicals) for 40 min. Then, cells were incubated with FITC-labeled goat anti-rabbit secondary antibody solution (1:100 dilution, #A0562, Beyotime, China) at room temperature for 1 h away from light.

Techniques: Phospho-proteomics, Activation Assay, Inhibition, Activity Assay, Western Blot, Flow Cytometry

Journal: Materials Today Bio

Article Title: Stiff matrix promotes lung cancer cell migration through down-regulating the Piezo1 channel expression to facilitate Ca 2+ -dependent filopodia formation

doi: 10.1016/j.mtbio.2026.102786

Figure Lengend Snippet: Schematic summary of the Piezo1/calcium/CaN-SSH/cofilin/filopodia formation pathway for substrate stiffness-induced cancer cell migration. Stiff matrix downregulates the expression of Piezo1 channel, which limits the [Ca 2+ ] i rise and the activity of CaN-SSH. Consequently, cofilin is phosphorylated and inactivated and thereby loses its potential to bind to and sever actin filaments, facilitates filopodia formation, and thereby promote cancer cell migration.

Article Snippet: Briefly, cells were harvested and fixed with 4 % paraformaldehyde for 30 min, then permeabilized with 0.3 % Triton X-100 for 10 min. After that, cells were incubated with the primary polyclonal antibody recognizing the extracellular domain of Piezo1 protein (1:100 dilution, #15939–1-AP, Proteintech, USA) or p-SSH1 (p Ser978) (1:100 dilution, #NBP3-23411, Novus Biologicals) for 40 min. Then, cells were incubated with FITC-labeled goat anti-rabbit secondary antibody solution (1:100 dilution, #A0562, Beyotime, China) at room temperature for 1 h away from light.

Techniques: Migration, Expressing, Activity Assay

Journal: bioRxiv

Article Title: Cluster nanoarchitecture and structural diversity of PIEZO1 in intact cells

doi: 10.1101/2024.11.26.625366

Figure Lengend Snippet: a , cryo-EM structure of PIEZO and localization of mGreenLantern(mGL)-tag (left) and cartoon illustrating the principle of MINFLUX/DNA-PAINT (right). DNA-PAINT utilizes complementary pairs of short oligonucleotides, an imager strand conjugated to the fluorophore and a docking strand conjugated to an sd-Nb that binds to the target. To determine the fluorophore position, the MINFLUX microscope performs an iterative 3D scanning procedure, which is repeated until the fluorophore is bleached or the imager strand dissociates from the target, such that ‘traces’ with multiple localization estimates of the same fluorophore are generated. b , shows a confocal scan of an N2a cell expressing PIEZO1-mGL and c , shows the corresponding MINFLUX localization data (left) and the distribution of the standard deviations of the MINFLUX traces along the x, y, and z-axis (right) d and e , show 3D-views, top views and side views of the raw MINFLUX localization data of the pit-shaped (d) and the spherical (e) clusters marked in c . For 360° rotation movies of the clusters shown in d and e, see Supplementary Video 1 and 2. f , Overlay of the 2D projections of the traces means of the top fifth and bottom four fifth of all pit-shaped clusters (blue circles, N=158 cluster from 19 cells) and spherical clusters (grey circles, N=93 clusters from 19 cells). Note, in pit-shaped clusters no channels are present at the center of the top fifth. g , bar graph showing the proportions of pit-shaped and spherical clusters.

Article Snippet: PIEZO1-mGreenLantern fusion protein (PIEZO1-mGL) was generated by amplifying the coding sequence of the green fluorescent protein mGreenLantern from a LifeAct-mGreenLantern plasmid (git from G. Petsko, Addgene #164459), and inserted with a SG linker after the C-ter of PIEZO1 plasmid, where the IRES-GFP has sequence has been excised beforehand by PCR.

Techniques: Cryo-EM Sample Prep, Microscopy, Generated, Expressing

Journal: bioRxiv

Article Title: Cluster nanoarchitecture and structural diversity of PIEZO1 in intact cells

doi: 10.1101/2024.11.26.625366

Figure Lengend Snippet: a , raw MINFLUX localization data color coded by Z-position of clusters imaged in control cells (top) and cells challenged with hypoosmotic solution (120 mOsm) before fixation (bottom). b , 3D-views, of the raw MINFLUX localization data of representative clusters from control (top) and osmotically stimulated (bottom) cells together with surface fits. For 360° rotation movies of the clusters see Supplementary Video 4-5. c , comparison of the cluster depths of control and hypo-osmotically stimulated cells (CTL: 144 ± 60 nm, N=158 vs. OSMO: 110.7 ± 54 nm, N=79, P=0.000052, two-tailed Student’s t-test). d , comparison of the proportions of pit-shaped (blue) and spherical clusters (grey) in control (CTL) and hypo-osmotically stimulated (OSMO) cells. The numbers provided in the bars represent the absolute number of clusters from the two categories. e , comparison of the mean ± sem number of PIEZO1 channel found in pit-shaped clusters. Circles represent the number of channels in individual clusters (CTL: 23.1±11.3, N=158 vs. OSMO: 18.7±8.0, N=79, P=0.0134, two-tailed Mann-Whitney test). f , histograms showing the distribution of the radii of all pit-shaped clusters measured at the pit opening. Medians are indicated by the red dashed lines. g , the left panel shows the side view of an example surface fit in which the contours of convex regions are depicted in blue and the contours of concave regions are shown in yellow. Gaussian curvatures of the fitted surfaces (see b) at the coordinates where channels were detected, were calculated and are shown as horizontal box plots. Boxes range from the lower quartile to the upper quartile, medians are shown as red lines and negative and positive curvatures are color coded in blue and yellow, respectively. Mean curvatures were compared using non-parametric Mann Whitney test (***, P = 0.0004, N=3550 for CTL and N=1358 for OSMO).

Article Snippet: PIEZO1-mGreenLantern fusion protein (PIEZO1-mGL) was generated by amplifying the coding sequence of the green fluorescent protein mGreenLantern from a LifeAct-mGreenLantern plasmid (git from G. Petsko, Addgene #164459), and inserted with a SG linker after the C-ter of PIEZO1 plasmid, where the IRES-GFP has sequence has been excised beforehand by PCR.

Techniques: Control, Comparison, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: Cluster nanoarchitecture and structural diversity of PIEZO1 in intact cells

doi: 10.1101/2024.11.26.625366

Figure Lengend Snippet: a , side and top views of PIEZO, in the curved (grey) and flattened (red) conformation. Distances between ALFA-tags from one trimer are designated as ‘interblade distance’ and the angles between the lines connecting the ALFA-tags (α,β,γ) as ‘interblade angles’. b , confocal image of an N2a cell expressing PIEZO1-ALFA-mGL (left) and top and side view of the corresponding MINFLUX localization data from the marked regions in the soma and neurites (Supplementary Video 6 for 360° rotation movies of the example shown here and Supplementary Video 7-10 for additional examples). c , 3D scatter plot of raw localizations of representative trimers found in a cell soma (top-left) and in a neurite (bottom-left). Localizations originating from fluorophores bound to different protomers are colored in black, dark grey and light grey. The 3D raw localizations were projected vertically onto the plane defined by the trace means (2D in-plane projection) and then fitted with a bivariate Gaussian distribution. The heatmaps in the right column show the probability densities of the Gaussian distributions. d , superparticle of all trimers found in somata (top) and in neurites (bottom), generated by aligning the trace means of each identified trimer to a reference trimer (equilateral triangle with a side length of 25nm) using the iterative closest point algorithm of Matlab. e , comparison of the mean ± sem interblade distances of trimers residing in somata (black bar, N=67 trimers from 10 cells) and neurites (grey bar, N=30 trimers from 10 cells) using two-tailed Students t-test (P=0.00019). Values from individual trimers are shown as grey circles. f , Interblade distance are plotted against neurite diameter, showing that there is no correlation between the two parameters (Pearson coefficient r = 0.153). g , cartoon showing the lack of an effect of membrane curvature on PIEZO1 conformation and highlighting differences in cytoskeletal architecture of somata and neurites.

Article Snippet: PIEZO1-mGreenLantern fusion protein (PIEZO1-mGL) was generated by amplifying the coding sequence of the green fluorescent protein mGreenLantern from a LifeAct-mGreenLantern plasmid (git from G. Petsko, Addgene #164459), and inserted with a SG linker after the C-ter of PIEZO1 plasmid, where the IRES-GFP has sequence has been excised beforehand by PCR.

Techniques: Expressing, Generated, Comparison, Two Tailed Test, Membrane

Journal: bioRxiv

Article Title: Cluster nanoarchitecture and structural diversity of PIEZO1 in intact cells

doi: 10.1101/2024.11.26.625366

Figure Lengend Snippet: a , cartoon showing the possible interplay of forces. PIEZO1 exerts a bending force onto the membrane (F PIEZO1 , F p ) while the membrane (F membrane , F m ) and the cytoskeleton (F cytoskeleton , F c ) exert opposing forces on PIEZO1, which work towards opening the channel. We hypothesize that the equilibrium of these forces eventually determines PIEZO1 conformation. The bottom panel illustrates the effect of cytochalasin-D on the actin cortex and the resulting change in the force equilibrium. b , example traces of PIEZO1-mediated currents evoked by negative pressure in cell-attached patch-clamp recordings. c , left panel shows normalized (I/Imax) pressure-response curves of PIEZO1 currents recorded from control (black circles) and cyto-D treated (red circles) cells (left panel). Comparison of the mean ± sem P50 values (P50 = pressure required for half-maximal activation, right panel) obtained by Boltzmann fits of the data shown in the left panel, using Student’s t-test (CTL: p50 = -40.6 ± 2.5 mmHg, N=20 vs. cyto-D: p50 = -29.1 ± 2.6 mmHg, N =16; P = 0.0038). White circles show P50 values from individual recordings. d , side views of MINFLUX raw localization data from a control (top) and a cyto-D-treated cell. Note, cyto-D treatment does not change the curvature of the cell-substrate interface. e , 2-D in-plane projections of the raw localization data (left) and heatmaps of the localization probability densities (right) of representative trimers from control (CTL, top) and cyto-D (bottom) treated cells. f , superparticle of all trimers found in control (CTL, top) and cyto-D (bottom) treated cells (see description in ). g , comparison of the mean ± sem interblade distances of all trimers from control (23.90 ± 5.14 nm, N=67, grey) and cyto-D (20.58 ± 5.30, N=53, red) treated cells using Students t-test (P=0.0007). Values from individual trimers are shown as grey circles.

Article Snippet: PIEZO1-mGreenLantern fusion protein (PIEZO1-mGL) was generated by amplifying the coding sequence of the green fluorescent protein mGreenLantern from a LifeAct-mGreenLantern plasmid (git from G. Petsko, Addgene #164459), and inserted with a SG linker after the C-ter of PIEZO1 plasmid, where the IRES-GFP has sequence has been excised beforehand by PCR.

Techniques: Membrane, Patch Clamp, Control, Comparison, Activation Assay