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