Journal: Communications Biology

Article Title: Nanosecond pulsed electric fields induce cell-size-dependent selective permeabilization of urothelial cancer cells

doi: 10.1038/s42003-025-09432-7

Figure Lengend Snippet: a A schematic of the setup that enables a fluorescence imaging of electroporated cell. Tungsten electrodes were positioned on the glass coverslip with seeded normal or cancer urothelial cells on the stable 45° angle between coverslip and electrodes. An 800 × 600 µm region of interest (ROI) was positioned such that its longer edge overlapped the line connecting the tips of the two electrodes. The ROI extended beyond the electrode gap on the side opposite the electrodes. ROI was divided into three equal subregions (subROI), each measuring 800 × 200 µm, by drawing two parallel lines, evenly spaced along the shorter axis of the ROI. b Calculated electric field distribution along the line perpendicular to the axis connecting the centers of both electrodes with 1 V applied between them. The ROI were placed in the electric field marked in ( c ). The time course of YP fluorescence measure as arbitrary units (a.u.) (mean ± the standard error of the mean (SEM)) in T24, UM-UC-3, SV-HUC-1 and HBLAK cells after electroporation by train of 200, 300-ns, 11.5 kV/cm at 10 Hz pulse in the solution with 1 µM YP and at different Ca 2+ concentration (marked in the titles) n = 18–20. The mean fluorescence was fitted with single-exponential curve (coefficients of determination (R²) > 0.95 for all tested conditions and cell lines). The comparison of area under curve (AUC) of YP fluoresce curves show highly significant differences between normal and cancer urothelial cells. Differences were assessed using one-way Welch’s ANOVA W (DFn; DFd). For 0 mM Ca²⁺: W(3, 36.07) = 56.15; for 2 mM Ca²⁺: W(3, 36.13) = 93.29; and for 5 mM Ca²⁺: W(3, 37.59) = 72.11. Dunnett’s T3 post hoc test was used to correct for multiple comparisons. Statistical significance is indicated as follows: ( p ≥ 0.05), * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. d Violin plot showing the distribution of mean YP florescence intensity at 180 seconds (s) after the exposure in 0 mM, 2 mM and 5 mM concentration of Ca 2+ n = 18–20. Differences were assessed using one-way Welch’s ANOVA followed by Dunnett’s T3 post hoc test for multiple comparisons: for 0 mM Ca²⁺: W(3, 35.06) = 16.22; for 2 mM Ca²⁺: W(3, 35.62) = 31.60; and for 5 mM Ca²⁺: W(3, 37.66) = 13.98.

Article Snippet: T24, UM-UC-3, RT4 and SV-HUC-1 cell line were acquired from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Fluorescence, Imaging, Electroporation, Concentration Assay, Comparison

Journal: Communications Biology

Article Title: Nanosecond pulsed electric fields induce cell-size-dependent selective permeabilization of urothelial cancer cells

doi: 10.1038/s42003-025-09432-7

Figure Lengend Snippet: a Schematic representation of membrane resealing dynamics, visualized through changes in YP fluorescence. (1). Prior to electroporation, the cell membrane remains intact, preventing the uptake of the membrane-impermeant dye YP. (2) Following electroporation, transient nanopores form in the plasma membrane, enabling the entry of YP and water from the extracellular environment into the cell. The kinetics of dye uptake can be fitted with a single-phase exponential curve, reflecting the dynamics of membrane permeability. (3) As the membrane reseals, dye uptake ceases and the fluorescence signal reaches a plateau. This plateau indicates successful membrane recovery, with no further entry of YP. b Time course of YP dye uptake in urothelial cancer cells and normal urothelial cells under three different extracellular Ca²⁺ concentrations: 0, 2, and 5 mM. The uptake dynamics were fitted with a single-phase exponential model, and the time constants τ (in s) are indicated next to each curve. The fits showed a strong correlation, with R² exceeding 0.95 for all cell lines under all tested conditions, indicating robust exponential uptake kinetics. Data are presented as mean ± SEM, with n = 18–20 per condition. c Average time constants derived from the exponential fits, shown as mean ± SEM ( n = 18–20). Points represent individual measurements from independent experimental replicates. Differences between different Ca 2+ concentration for each cell line and electric field intensity were assessed using one-way Welch’s ANOVA for followed by Dunnett’s T3 post hoc test for multiple comparisons. Welch’s ANOVA results (W (DFn, DFd)) for comparisons across Ca²⁺ concentrations at 11.5 kV/cm, 10 kV/cm, and 7.5 kV/cm were as follows: For HBLAK: W(2.000, 35.04) = 1.125; W(2.000, 32.86) = 0.5627; W(2.000, 33.46) = 0.6787. For SV-HUC-1: W(2.000, 32.85) = 0.5254; W(2.000, 32.32) = 1.099; W(2.000, 33.62) = 1.085. For T24: W(2.000, 37.64) = 6.939; W(2.000, 36.92) = 14.82; W(2.000, 28.14) = 22.13. For UM-UC-3: W(2.000, 36.72) = 6.800; W(2.000, 36.92) = 14.82; W(2.000, 37.80) = 2.404.

Article Snippet: T24, UM-UC-3, RT4 and SV-HUC-1 cell line were acquired from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Membrane, Fluorescence, Electroporation, Clinical Proteomics, Permeability, Derivative Assay, Concentration Assay

Journal: Communications Biology

Article Title: Nanosecond pulsed electric fields induce cell-size-dependent selective permeabilization of urothelial cancer cells

doi: 10.1038/s42003-025-09432-7

Figure Lengend Snippet: a Representative images of monolayers of HBLAK, SV-HUC-1, UM-UC-3, and T24 cells stained with Phalloidin and Hoechst 33342 dyes to visualize actin filaments (red) and nuclei (blue), respectively. b Morphological features of the cells area measurements were obtained from 18 randomly selected ROIs (each 250 µm × 250 µm) containing approximately 40–80 stained cells. The average of measured morphological feature of cells within each ROI was treated as an independent data point for statistical analysis, resulting in n = 18. Data were visualized using violin plots, and statistical comparisons between cell lines were performed using Welch’s ANOVA, followed by Dunnett’s T3 post hoc testing. Welch’s ANOVA revealed statistically significant differences between cell lines in average cell area (W(3, 37.28) = 46.01), major axis length (W(3, 37.46) = 38.88), and minor axis length (W(3, 37.32) = 49.28). No significant differences were observed in the form factor (W(3, 36.34) = 0.98). c Plot of the electroporation threshold electric field as a function of pulse duration for cells of varying radii. The external electric field (E, in V/m) necessary to reach electroporation threshold was calculated for the cathode-facing pole of a spherical cell, assuming a membrane charging time constant (τ) of 2 µs. See main text for methodological details. d Representative images and violin plots showing the average cell area and form factor of single cells across 9 tissue microarrays (TMAs), with each point representing the mean value per TMA ( n = 9). The dataset includes 47 normal urothelial tissue samples, 66 samples from primary urothelial carcinomas, and 56 samples from lymph node metastases. Statistical comparison of mean urothelial cell area across normal urothelial tissue, urothelial carcinoma, and lymph node metastasis was performed using weighted one-way ANOVA, with the number of tissue sections per array used as weights and the Tukey adjustment for multiple comparisons. Pairwise comparisons using weighted marginal means revealed significantly larger mean cell areas in normal urothelial tissue compared to both cancer and lymph node tissue (F(2, 24) = 13.42) as well as lower form factor of normal urothelial cells, (F(2, 24) = 6.39).

Article Snippet: T24, UM-UC-3, RT4 and SV-HUC-1 cell line were acquired from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Staining, Electroporation, Membrane, Comparison

Journal: Communications Biology

Article Title: Nanosecond pulsed electric fields induce cell-size-dependent selective permeabilization of urothelial cancer cells

doi: 10.1038/s42003-025-09432-7

Figure Lengend Snippet: a Spheroids and PDOs were positioned between two stainless steel electrodes. YP uptake was monitored via fluorescence imaging for 10 min post-exposure to nsPEFs. Fluorescence images were pseudocolored to enhance visualization. b Representative pseudocolored images of spheroids and PDOs before and after nsPEFs exposure. c Time-course analysis of YP fluorescence uptake in spheroids derived from normal urothelial cells and urothelial cancer cells, under different extracellular Ca²⁺ concentrations. The spheroids were observed for 10 min following exposure. Data were shown as mean ± SEM ( n = 9–11). Statistical comparisons of the AUC of fluorescence between normal urothelial SV-HUC-1 spheroids and cancer RT4 spheroids with PDO were performed using Welch’s ANOVA, followed by Dunnett’s T3 post hoc test for multiple comparisons: for 0 mM Ca²⁺, W(4, 28.14) = 28.34, for 2 mM Ca²⁺, W(4, 20.46) = 38.53 and for 5 mM Ca²⁺, W(4, 18.09) = 53.60. d Quantification and correlation of single-cell size in PDOs and YP fluorescence after exposure. The mean cell size was calculated based on H&E-stained slides of spheroids. Statistical comparisons of single cell area between spheroids and PDOs were performed using Welch’s ANOVA, followed by Dunnett’s T3 post hoc test for multiple comparisons (W(4, 787.4) = 10.74). The correlation between mean single-cell size in spheroids and YO-PRO-1 fluorescence intensity 10 min after nsPEF exposure in 2 mM Ca²⁺ was assessed by linear regression analysis. The analysis included data from 45 spheroids and PDOs derived from RT4, SV-HUC-1, PDO 154, PDO 270, and PDO 319. Each dot represents a single spheroid ( n = 45).

Article Snippet: T24, UM-UC-3, RT4 and SV-HUC-1 cell line were acquired from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Fluorescence, Imaging, Derivative Assay, Staining

Journal: Communications Biology

Article Title: Nanosecond pulsed electric fields induce cell-size-dependent selective permeabilization of urothelial cancer cells

doi: 10.1038/s42003-025-09432-7

Figure Lengend Snippet: a Changes in the 2D projected area of spheroids and PDOs (mean ± SEM, n = 9–11) following nsPEFs exposure in solutions containing 0 mM, 2 mM, or 5 mM Ca²⁺. Statistical comparisons of the area under the curve (AUC) for 2D projected area changes over time were performed using Welch’s ANOVA, followed by Dunnett’s T3 post hoc test to compare normal urothelial SV-HUC-1 spheroids with cancer RT4 spheroids and PDOs in different Ca 2+ concentrations: for 0 mM Ca²⁺, W(4, 21.73) = 835.0; for 2 mM Ca²⁺, W(4, 19.38) = 485.5; and for 5 mM Ca²⁺, W(4, 19.46) = 326.2. b Mechanical properties of spheroids of normal SV-HUC-1 and cancer RT4 cells were evaluated using AFM force mapping, conducted at 25 points (5 × 5 grid) across the spheroid surface at defined time intervals post-exposure. Young’s modulus values were calculated from force–distance curves by fitting them to the Hertz model. Values obtained from a single spheroid were averaged to yield one representative value per spheroid. c Comparison of Young’s modulus (“stiffness”) between SV-HUC-1 and RT4 spheroids in 0 mM and 2 mM Ca²⁺ solutions, both before and 10 minutes after exposure to nsPEFs ( n = 9–11). Data are presented as mean ± SEM. Statistical comparisons were performed using two-way ANOVA with Geisser–Greenhouse correction for unequal variability: Before exposure: effect of Ca²⁺, F(1, 32) = 3.315; effect of cell line, F(1, 32) = 5.963. After exposure: effect of Ca²⁺, F(1, 34) = 0.104; effect of cell line, F(1, 34) = 23.24. d Young’s modulus values were normalized to the pre-exposure baseline and the mean values were fitted using a single-exponential nonlinear regression model (mean ± SEM, n = 9–11): R² for the exponential fits were 0.85 and 0.99 for SV-HUC-1 spheroids, and 0.95 and 0.94 for RT4 spheroids under 0 mM and 2 mM Ca²⁺ conditions, respectively. Points represent individual measurements from independent experimental replicates. For statistical comparisons at defined time points, repeated measures two-way ANOVA with Geisser–Greenhouse correction was applied, followed by Šidák’s multiple comparisons test. In 0 mM Ca²⁺: effect of time, F(2.235, 33.53) = 48.26; effect of cell type, F(1, 15) = 15.22. In 2 mM Ca²⁺: effect of time, F(1.665, 26.65) = 55.19; effect of cell type, F(1, 16) = 3.252.

Article Snippet: T24, UM-UC-3, RT4 and SV-HUC-1 cell line were acquired from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Comparison

Journal: Journal of Translational Medicine

Article Title: Urothelial Piezo1 channel contributes to BOO-induced inflammation, EMT, and bladder fibrosis via activation of NLRP3 inflammasome

doi: 10.1186/s12967-026-07688-z

Figure Lengend Snippet: Knockout of urothelial Piezo1 attenuated BOO (8 weeks)-induced bladder fibrosis and dysfunction in mice. ( A-B ) Comparison of the bladder weight of mice in the three groups (sham, BOO, and BOO in Piezo1 -KO mice (BOO+KO)), showing that BOO induced a smaller increase in bladder weight in Piezo1-KO mice than in their littermate controls. The surgeries in the sham and BOO groups were conducted in Piezo1 -KO control mice. Bladders were harvested at 8 weeks after BOO. n = 5 mice in each group in summary data (B). ( C-D ) Masson’s trichrome staining of the bladder tissues showing that BOO induced a lesser extent of bladder fibrosis in Piezo1-KO mice than in control mice. Scale bar in (C): 500 μm. The ratio of collagen in the whole bladder was measured and summarized in (D), n = 5 in each group. ( E-F ) WB analysis confirmed the BOO (8 weeks)-induced increase in fibronectin and collagen I in the bladder mucosa. WB also showed that BOO induced a decrease in E-cadherin levels and an increase in N-cadherin and α-SMA levels, indicating the presence of EMT. However, all these changes were attenuated in Piezo1-KO mice ( n = 4 in each group). ( G-H ) Representative traces and the summary data of cystometrogram (CMG) recordings showing that BOO induced a decreased inter-voiding interval (IVI) and reduced bladder compliance, which were significantly improved in Piezo1-KO mice. However, Piezo1 knockout did not alter the BOO-induced increase in voiding pressure ( n = 5 in each group). Piezo1-KO refers to Upk2Cre +/– Piezo1 flox/flox mice; Piezo1-KO control refers to Upk2Cre –/– Piezo1 flox/flox mice. * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the sham group. & p < 0.05, && p < 0.01, &&& p < 0.001 compared with the BOO group

Article Snippet: A Piezo1-knockout SV-HUC-1 cell line, in which the endogenous Piezo1 channel was deleted using CRISPR/Cas9 technology, was purchased from OBiO Technology (Shanghai, China).

Techniques: Knock-Out, Comparison, Control, Staining

Journal: Journal of Translational Medicine

Article Title: Urothelial Piezo1 channel contributes to BOO-induced inflammation, EMT, and bladder fibrosis via activation of NLRP3 inflammasome

doi: 10.1186/s12967-026-07688-z

Figure Lengend Snippet: Activation of Piezo1 results in EMT and profibrotic changes in urothelial cells in vitro. WB experiments showed that stimulation of SV-HUC-1 cells with Yoda1( A ) (10 μM for 24 hours) or cyclic high pressure ( B ) (100 cmH₂O for 5 min every 2 hours for 24 hours) induced increased protein expression of fibronectin, collagen I, N-cadherin, and α-SMA and decreased the protein expression of E-cadherin in SV-HUC-1 cells. By contrast, these changes were less in Piezo1-KO SV-HUC-1 cells ( n = 4 in each group). * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the NC group. & p < 0.05, && p < 0.01, &&& p < 0.001 compared with the NC+Yoda1or high pressure group

Article Snippet: A Piezo1-knockout SV-HUC-1 cell line, in which the endogenous Piezo1 channel was deleted using CRISPR/Cas9 technology, was purchased from OBiO Technology (Shanghai, China).

Techniques: Activation Assay, In Vitro, Expressing

Journal: Journal of Translational Medicine

Article Title: Urothelial Piezo1 channel contributes to BOO-induced inflammation, EMT, and bladder fibrosis via activation of NLRP3 inflammasome

doi: 10.1186/s12967-026-07688-z

Figure Lengend Snippet: Piezo1 activation induced increased expression of NF-κB, NLRP3, ASC, caspase-1, and IL-1β in vivo and in vitro. ( A ) WB analysis of protein expressions of NF-κB, p-NF-κB, NLRP3, caspase-1, cleaved caspase-1(Cl-caspase-1), pro-IL-1β, and ASC in the bladder mucosa of the sham, BOO, and BOO+KO groups, showing that BOO (2 weeks) induced increased expression of these proteins, which were significantly attenuated in Piezo1-KO mice. IL-1β level in the bladder mucosa was detected by ELISA. Data are shown as mean ± SEM ( n = 4 in each group). * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the sham group. & p < 0.05, && p < 0.01, &&& p < 0.001 compared with the BOO group. ( B-C ) WB analysis of the expression of the aforementioned proteins in cultured SV-HUC-1 cells showing that treatment with Yoda1 (B) or cyclic high pressure (C) to activate Piezo1 increased the expression of the aforementioned proteins, whereas the increase in the expression of these proteins was not prominent in Piezo1-KO SV-HUC-1 cells. Yoda1 and pressure treatments were the same as those in Fig. . IL-β levels in cell supernatants were measured by ELISA. Data are shown as mean ± SEM ( n = 4 in each group). * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the NC group. & p < 0.05, && p < 0.01, &&& p < 0.001 compared with the NC+Yoda1or high pressure group

Article Snippet: A Piezo1-knockout SV-HUC-1 cell line, in which the endogenous Piezo1 channel was deleted using CRISPR/Cas9 technology, was purchased from OBiO Technology (Shanghai, China).

Techniques: Activation Assay, Expressing, In Vivo, In Vitro, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Journal of Translational Medicine

Article Title: Urothelial Piezo1 channel contributes to BOO-induced inflammation, EMT, and bladder fibrosis via activation of NLRP3 inflammasome

doi: 10.1186/s12967-026-07688-z

Figure Lengend Snippet: Activation of Piezo1 results in Ca 2+ increase and ATP release. ( A ) assessment of intracellular Ca 2+ showed that stimulation with Yoda1 (10 μM for 24 hours) or cyclic high pressure (100 cmH₂O for 5 min every 2 hours for 24 hours) induced a significant increase in baseline intracellular Ca 2+ in SV-HUC-1 cells compared with that in Piezo1-KO SV-HUC-1 cells ( n = 3 in each group). Scale bar: 50 μm. *** p < 0.001 compared with the NC group, &&& p < 0.001 compared with the NC+Yoda1or high pressure group. ( B ) ATP measurement indicated that in response to stimulation with Yoda1 or high pressure, SV-HUC-1 cells produced a significantly higher ATP release, whereas in Piezo1-knockout SV-HUC-1 cells ATP release was attenuated ( n = 3 in each group). *** p < 0.001 compared with the NC group, &&& p < 0.001 compared with the NC+Yoda1or high pressure group. ( C ) ATP measurement in the urine indicates that the urinary ATP level was increased in BOO (2 weeks) mice compared with that in the control, and it was attenuated in Piezo1-KO mice ( n = 5 in each group). *** p < 0.001 compared with the sham group. &&& p < 0.001 compared with the BOO group

Article Snippet: A Piezo1-knockout SV-HUC-1 cell line, in which the endogenous Piezo1 channel was deleted using CRISPR/Cas9 technology, was purchased from OBiO Technology (Shanghai, China).

Techniques: Activation Assay, Produced, Knock-Out, Control

Journal: Journal of Translational Medicine

Article Title: Urothelial Piezo1 channel contributes to BOO-induced inflammation, EMT, and bladder fibrosis via activation of NLRP3 inflammasome

doi: 10.1186/s12967-026-07688-z

Figure Lengend Snippet: Activation of Piezo1 results in ROS production. ( A ) Assessment of the intracellular ROS level with the fluorescence probe DCFH-DA showed that Yoda1 or cyclic high pressure stimulation induced a significant increase in intracellular ROS in SV-HUC-1 cells, whereas the ROS increase was not evident in Piezo1-KO SV-HUC-1 cells ( n = 3 in each group). Scale bar: 100 μm. *** p < 0.001 compared with the NC group, &&& p < 0.001 compared with the NC+Yoda1or high pressure group. ( B ) ROS assessment with DHE reveal that BOO (2 weeks) induced a significant increase in ROS in the bladder mucosa and the increase was not evident in Piezo1-KO mice ( n = 5 in each group). Scale bar: 100 μm. *** p < 0.001 compared with the sham group. &&& p < 0.001 compared with the BOO group

Article Snippet: A Piezo1-knockout SV-HUC-1 cell line, in which the endogenous Piezo1 channel was deleted using CRISPR/Cas9 technology, was purchased from OBiO Technology (Shanghai, China).

Techniques: Activation Assay, Fluorescence

Journal: Journal of Translational Medicine

Article Title: Urothelial Piezo1 channel contributes to BOO-induced inflammation, EMT, and bladder fibrosis via activation of NLRP3 inflammasome

doi: 10.1186/s12967-026-07688-z

Figure Lengend Snippet: Activation of Piezo1 results in increased expression of TGF-β1. ( A-B ) WB analysis of the expression of the aforementioned proteins in culture SV-HUC-1 cells showing that treatment with Yoda1 (A) or cyclic high pressure (B) increased the expression of the aforementioned proteins, whereas, the increase in the expression of these proteins was not prominent in Piezo1-KO SV-HUC-1 cells. Yoda1 and pressure treatments were same as those in Fig. . Data are shown as mean ± SEM ( n = 4 in each group). * p < 0.05, ** p < 0.01 compared with the NC group. & p < 0.05, && p < 0.01 compared with the NC+Yoda1or high pressure group. ( C ) WB analysis of protein expression of TGF-β1 and smad3 in the bladder mucosa from the sham, BOO, and BOO+KO groups, showing BOO (2 weeks) induced increased expression of these proteins, which were significantly attenuated in Piezo1-KO mice ( n = 4 in each group). * p < 0.05, ** p < 0.01 compared with the sham group. & p < 0.05 compared with the BOO group

Article Snippet: A Piezo1-knockout SV-HUC-1 cell line, in which the endogenous Piezo1 channel was deleted using CRISPR/Cas9 technology, was purchased from OBiO Technology (Shanghai, China).

Techniques: Activation Assay, Expressing