Fig 2: Direct current electric fields regulate PDLIM7 polarization and promote breast cancer cell migration. (A) Schematic cross‐section of the electro‐bioreactor. (B) Representative fluorescence images showing the distribution of F‐actin and PDLIM7 in cells subjected to no current, low current (200 mV/mm), and high current (400 mV/mm) stimulation for 1 hour. Green: FITC‐labeled F‐actin; Red: TRITC‐labeled PDLIM7. Scale bar: 10 µm. (C) Top: Time‐lapse FRET images of MDA‐MB‐231 cells expressing the PDLIM7‐M‐cpstFRET probe under a 400 mV/mm electric field for 15 min. Bottom: Normalized CFP/FRET ratios at the cell front and rear (mean ± SD, n ≥ 6 cells). Scale bar: 10 µm. Calibration bar: 0.00–1.30. (D,E) Transwell invasion assay of MDA‐MB‐231 cells transfected with pCMV‐PDLIM7 plasmid or PDLIM7‐siRNA. Quantification of invaded cells (mean ± SD, n = 6). Statistical analysis: unpaired Student's t‐test (* P < 0.05, ** P < 0.01). (F, G) Dissemination of cells from spheroids embedded in 3D collagen gels, transfected as indicated, at 0 and 24 h. Quantification of cell expansion (mean ± SD, n = 6). Unpaired Student's t‐test (** P < 0.01). (H) Forward migration velocity (µm/min) during electrotaxis over 1 h (mean ± SEM, n ≥ 15 cells). One‐way ANOVA was used for comparisons (*** P < 0.001, **** P < 0.0001). (I) Top: Representative images of Lifeact‐transfected cells during electrotaxis (400 mV/mm). Bottom: Rose plots showing orientation angles; bar magnitudes indicate the fraction of cells in each trajectory angle bin (n ≥ 10 cells). Scale bar: 10 µm. (J) Fluorescence images of F‐actin (green, Phalloidin) and nuclei (blue, DAPI) in MDA‐MB‐231 cells. Scale bar: 10 µm.

Fig 3: PDLIM7 Ser190 phosphorylation promotes TNBC invasion in vitro and in vivo. (A) Dissemination of MDA‐MB‐231 spheroids transfected with PDLIM7‐WT, S190A, S190E, or S144A constructs embedded in 3D collagen gels at 0 and 24 h under 400 mV/mm stimulation. Scale bar: 100 µm. (B) Quantification of cell expansion (mean ± SD, n = 6). One‐way ANOVA (** P < 0.01, *** P < 0.001). (C) Representative bioluminescence images of systemic metastases in nude mice at 0 and 28 days post‐injection. (D) Quantification of fluorescence intensity in tumor regions (mean ± SD, n = 6). One‐way ANOVA (** P < 0.01, *** P < 0.001). (E) Orthotopic implantation assay showing CT images. (F) Representative lung tissues, and H&E‐stained lung sections. Red circles indicate metastatic nodules on the 28th day.

Fig 4: TMEM16A and SK regulate osmotic pressure asymmetry during electrotactic migration. (A) Left: Immunofluorescence images of TMEM16A and SK (KCNN3) under 400 mV/mm stimulation. Green: FITC‐TMEM16A; Red: TRITC‐PDLIM7. Scale bar: 10 µm. Right: Front‐to‐rear intensity ratios. Unpaired Student's t‐test (*** P < 0.001, **** P < 0.0001). (B) Cell volume measurements after TMEM16A or SK inhibition under electric field (mean ± SD, n ≥ 6). One‐way ANOVA (* P < 0.05, **** P < 0.0001, ns: not significant). (C) Up: Membrane potential dye images post‐inhibitor treatment. Bottom: Quantification of membrane potential changes at MDA‐MB‐231 cells front and rear. Unpaired Student's t‐test (*** P < 0.001, ns: not significant). Scale bar: 10 µm. (D) Quantification of membrane potential changes (mean ± SD, n ≥ 6). Unpaired Student's t‐test (*** P < 0.001, ns: not significant). (E, G) Current traces of TMEM16A and SK channels post‐inhibitor treatment. (F,H) Quantification of TMEM16A and SK current density. (mean ± SD, n ≥ 6). Unpaired Student's t‐test (*** P < 0.001). (I,K) Time‐lapse imaging of K⁺ and Cl− under inhibitor treatment (mean ± SD, n ≥ 6). Scale bar: 10 µm. (J,L) Quantification of K⁺ and Cl− fluorescence intensity at MDA‐MB‐231 cells front and rear after treatment with control, electric field and inhibitor. (mean ± SEM, n ≥ 6 cells). Unpaired Student's t‐test (ns: not significant, *** P < 0.001, **** P < 0.0001). (M) Up: FRET images of Vimentin‐M‐cpstFRET expressing cells post‐inhibitor treatment. Bottom: Normalized CFP/FRET ratios at MDA‐MB‐231 cells front and rear. Unpaired Student's t‐test (*** P < 0.001, ns: not significant). Scale bar: 10 µm. Calibration bar: 0.00–1.30. (N) Quantified front‐to‐rear tension ratios (mean ± SD, n ≥ 6). Unpaired Student's t‐test (*** P < 0.001, ns: not significant).

Fig 5: Ser190 phosphorylation in the IDR domain regulates PDLIM7 tension. (A) Left: AlphaFold2‐predicted structure of PDLIM7 binding to PKA. Orange: PDLIM7 IDR domain; Green: PKA. Right: Enlarged view of the binding interface with affinity values. (B) Immunoprecipitation showing endogenous PKA interaction with PDLIM7 in fresh human TNBC tissues. (C) Representative FRET images of MDA‐MB‐231 cells expressing PDLIM7‐M‐cpstFRET or PDLIM7△IDR‐M‐cpstFRET probes. Scale bar: 10 µm. (D) FRET images of cells expressing PDLIM7 variants with specific Ser/Ala or Ser/Glu mutations (S95A, S109A, S144A, S190A, S190E) under 400 mV/mm stimulation. Scale bar: 10 µm. Calibration bar: 0.00–1.30. (E) Quantification of normalized CFP/FRET ratios (mean ± SEM, n ≥ 20 cells). One‐way ANOVA (*** P < 0.001, **** P < 0.0001). (F) Western blot analysis of phosphorylated PDLIM7 in cells expressing WT or mutant GFP‐PDLIM7 constructs. (G) Quantification of phosphorylated PDLIM7/input ratio (n ≥ 3). One‐way ANOVA (* P < 0.05). (H) Representative FRET images of cells treated with PKA inhibitors/activators under 400 mV/mm stimulation. Scale bar: 10 µm. Calibration bar: 0.00–1.30. (I) Quantification of normalized CFP/FRET ratios (mean ± SEM, n ≥ 6 cells). One‐way ANOVA (* P < 0.05, *** P < 0.001). (J) Immunofluorescence of endogenous PKA and PDLIM7 in cells exposed to 0, 200, or 400 mV/mm fields. Green: FITC‐labeled PDLIM7; Red: TRITC‐labeled PKA. Scale bar: 10 µm.