Journal: Journal of Biological Physics

Article Title: Estradiol alters actin and protrusion dynamics in endometriotic epithelial cells

doi: 10.1007/s10867-026-09712-1

Figure Lengend Snippet: Supplementary file2 3D rendering of a 12Z cell’s actin dynamics. 12Z cells were transfected with a LifeAct-GFP plasmid via nucleofection and imaged over time using lattice lightsheet microscopy. The movie depicts a 3D rendering of a 12Z cell (the same cell as in Movie Supplemental_2D.avi) exhibiting rapid protrusion dynamics. Total time elapsed = 1 hour (MP4 4077 KB)

Article Snippet: The 12Z cell line was purchased from Applied Biological Materials (Cat. #T0764).

Techniques:

Journal: Journal of Biological Physics

Article Title: Estradiol alters actin and protrusion dynamics in endometriotic epithelial cells

doi: 10.1007/s10867-026-09712-1

Figure Lengend Snippet: Experimental setup. a 12Z cells were transfected with a LifeAct-GFP plasmid via nucleofection (an electroporation-based method) and imaged over time using volumetric lattice lightsheet microscopy, which employs long thin beams to illuminate the sample with subcellular resolution. b A maximum intensity projection showing that the boundary of the actin cytoskeleton (LifeAct-GFP) coincides with the plasma membrane. c An example output maximum intensity projection from a lattice lightsheet microscopy time-lapse of a cell transfected with LifeAct-GFP with frames from t = 0, 1 min, 5 min, 10 min, and 60 min. Scalebars = 20 µm

Article Snippet: The 12Z cell line was purchased from Applied Biological Materials (Cat. #T0764).

Techniques: Transfection, Plasmid Preparation, Electroporation, Microscopy, Clinical Proteomics, Membrane

Journal: Journal of Biological Physics

Article Title: Estradiol alters actin and protrusion dynamics in endometriotic epithelial cells

doi: 10.1007/s10867-026-09712-1

Figure Lengend Snippet: 3D isosurfaces of 12Z cells obtained from lattice lightsheet imaging, with some notable cellular features ( a-d ) and representative time series ( e–g ). a Some cells exhibited actin waves at the leading edge of the cell and hair-like protrusions at the lagging edge of the cell (indicated by white arrows). Several cells also exhibited membrane ruffling ( b ), 3D filopodial extensions ( c , d ), and active protrusions ( d ). The dynamics of the membrane ruffling and lamellipodia growth were captured ( e ). Protrusion retraction ( f, g ) and formation ( f ) were also visualized. All scalebars = 20 µm

Article Snippet: The 12Z cell line was purchased from Applied Biological Materials (Cat. #T0764).

Techniques: Imaging, Membrane

Journal: Journal of Biological Physics

Article Title: Estradiol alters actin and protrusion dynamics in endometriotic epithelial cells

doi: 10.1007/s10867-026-09712-1

Figure Lengend Snippet: Effect of E2 on 12Z shape and morphodynamics after 15 min (N = 7 cells per group) or 24 h (N = 5 cells per group) of treatment incubation. Circularity (a) , rate of change in circularity (b) , solidity (c) , and rate of change in solidity (d) over the duration of each time-lapse were extracted from binarized maximum intensity projections, and the means ± SD are plotted. * P ≤ 0.05, ** P ≤ 0.01

Article Snippet: The 12Z cell line was purchased from Applied Biological Materials (Cat. #T0764).

Techniques: Incubation

Journal: Journal of Biological Physics

Article Title: Estradiol alters actin and protrusion dynamics in endometriotic epithelial cells

doi: 10.1007/s10867-026-09712-1

Figure Lengend Snippet: Impact of E2 on actin optical flow alignment in 12Z cells. a Optical flow (OF) was calculated from actin fluorescence for all timepoints to determine how the actin is moving within the video. The optical flow vectors are displayed on top of one of the corresponding actin fluorescence images. b The actin optical flow alignment (OF alignment) is displayed for the cell in ( a ). Higher values of optical flow alignment mean that the optical flow in that region is pointing in the same direction (see Materials and methods). Using the binarized masks from Fig. 5 the average optical flow alignment was found inside the protrusions and the cell body. The mean protrusion optical flow alignment (c) and mean ratio of protrusion optical flow alignment to cell body optical flow alignment ( d ) are plotted ± SD, representing individual cell data ( N = 5–7 cells per group). * P ≤ 0.05, ** P ≤ 0.01

Article Snippet: The 12Z cell line was purchased from Applied Biological Materials (Cat. #T0764).

Techniques: Fluorescence

Journal: bioRxiv

Article Title: Fibrotic substrate stiffness enhances endometriotic epithelial cell motility

doi: 10.64898/2025.12.31.697209

Figure Lengend Snippet: 12Z cells were cultured on substrates of varying stiffness overnight before assessing their morphology. A) Phase contrast images were taken of 12Zs on 2 kPa, 30 kPa, and glass substrates. We quantified the cell B) area, C) circularity, D) solidity, and E) ratio of the minor to major axis. Scalebars = 100 µm. Data points represent trials; error bars represent standard error of the mean. 30-40 cells were analyzed per stiffness per trial. * P ≤ 0.05.

Article Snippet: The 12Z human endometriotic epithelial cell line was purchased from Applied Biological Materials (Cat. # T0764).

Techniques: Cell Culture

Journal: bioRxiv

Article Title: Fibrotic substrate stiffness enhances endometriotic epithelial cell motility

doi: 10.64898/2025.12.31.697209

Figure Lengend Snippet: 12Z cells were cultured on substrates of varying stiffness overnight before measuring their random motility using timelapse microscopy for 12 hr. A) Trajectories of individual cells are plotted across all three trials. We quantified B) percentage of cells categorized as fast (speed>1 µm/min), medium (0.5 µm/min

Article Snippet: The 12Z human endometriotic epithelial cell line was purchased from Applied Biological Materials (Cat. # T0764).

Techniques: Cell Culture, Microscopy

Journal: bioRxiv

Article Title: Fibrotic substrate stiffness enhances endometriotic epithelial cell motility

doi: 10.64898/2025.12.31.697209

Figure Lengend Snippet: 12Z spheroids were seeded on substrates of varying stiffness and their outgrowth was measured using timelapse microscopy for 20 hr. A) Phase contrast images show spheroids on 2 kPa, 30 kPa, or glass substrates at t = 0 h, t = 2.5 hr, and t = 20 hr. We quantified B) the fold increase in projected spheroid area at multiple time points. We provide a zoomed in view of the fold change in projected area at C) t = 2.5 hr and D) t = 5 hr. We also measured the speed of the leading edge of the spheroid, averaging across E) the whole 20-hr timelapse, F) the first 2.5 hr, and G) the first 5 hr. Scalebars = 100 µm. Data points represent spheroids pooled across three trials; error bars represent standard error of the mean. 10-15 spheroids were analyzed per stiffness. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.

Article Snippet: The 12Z human endometriotic epithelial cell line was purchased from Applied Biological Materials (Cat. # T0764).

Techniques: Microscopy

Journal: bioRxiv

Article Title: Fibrotic substrate stiffness enhances endometriotic epithelial cell motility

doi: 10.64898/2025.12.31.697209

Figure Lengend Snippet: 12Z cells were cultured on substrates overnight prior to fixation and fluorescence microscopy. Confocal images show F-actin (Phalloidin), focal adhesion proteins (paxillin), and nuclei (Hoechst) for cells on 2 kPa, 30 kPa, and glass substrates. We quantified B) the fraction of cells exhibiting actin stress fibers, and C) the fraction of stress fibered cells exhibiting more than 5 stress fibers, D) the fraction of cells exhibiting punctate actin membrane structures, E) the average number of focal adhesions per cell, and F) the focal adhesion size. Scalebars = 50 µm. Data points represent trials; error bars represent standard error of the mean. 4-5 images were analyzed per stiffness per trial. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. FA; focal adhesion.

Article Snippet: The 12Z human endometriotic epithelial cell line was purchased from Applied Biological Materials (Cat. # T0764).

Techniques: Cell Culture, Fluorescence, Microscopy, Membrane

Journal: bioRxiv

Article Title: Fibrotic substrate stiffness enhances endometriotic epithelial cell motility

doi: 10.64898/2025.12.31.697209

Figure Lengend Snippet: 12Z cells were cultured on 2 kPa and 30 kPa substrates overnight before contractility modulators––LPA and Y27632, respectively––were added and cell motility was measured via timelapse microscopy for 4 hr. A) Phase contrast images show 12Zs cultured on 2 kPa and 30 kPa after ∼2 hr of treatment with the vehicle or drug. B) Trajectories of individual cells are plotted across all trials. We quantified C) average cell speed, D) directional persistence, and E) cell speed over time, where each data point represents an average from a subset of 12 cells (3 cells per trial). Scalebars = 100 µm. Unless specified, data points represent trials; error bars represent standard error of the mean. 30-40 cells were analyzed per stiffness per trial. * P ≤ 0.05, ** P ≤ 0.01. CTRL; vehicle control. LPA; lysophosphatidic acid.

Article Snippet: The 12Z human endometriotic epithelial cell line was purchased from Applied Biological Materials (Cat. # T0764).

Techniques: Cell Culture, Microscopy, Control

Journal: The FASEB Journal

Article Title: S1P 3 Receptor Mediates the Proinflammatory Effect of the Endocannabinoid 2‐Arachidonoylglycerol in Endometriotic Epithelial Cells

doi: 10.1096/fj.202502415R

Figure Lengend Snippet: Cannabinoid receptors and enzymes are expressed in human endometriotic lesions. (A) qPCR analysis was performed using TaqMan Gene Expression Assay probes specific for cannabinoid receptors CB1, CB2, GPR18, and TRPV1 in the endometrium of healthy women (HE) ( n = 15) or endometriotic lesions (EL) of different localization ( n = 20: 7 ovarian endometriosis and 13 deep infiltrating endometriosis). Results were analyzed employing the 2 −ΔCt method. (B) Representative immunohistochemical images of CB1, CB2, GPR18 and TRPV1 expression in endometriotic lesions ( n = 15: 11 ovarian endometriosis and 4 deep infiltrating endometriosis). The staining with DAB produced a brown precipitate at the site of antibody binding, localized both in epithelial (black arrows) and stromal (red arrows) cells of the endometriotic lesions. The negative control was obtained by processing tissue sections in parallel with the same IHC protocol, omitting the primary antibody. Magnification: ×40, scale bar: 30 μm. (C) qPCR analysis was performed using TaqMan Gene Expression Assay probes specific for cannabinoid enzymes NAPE‐PLD, DAGL, FAAH, and MAGL in the endometrium of healthy women (HE) ( n = 15) or endometriotic lesions (EL) of different localization ( n = 20: 7 ovarian endometriosis and 13 deep infiltrating endometriosis). Results were analyzed employing the 2 −ΔCt method. NAPE‐PLD and MAGL mRNA levels are significantly increased in EL compared to HE (Student's t ‐test *p < 0.05).

Article Snippet: Human endometriotic epithelial 12Z cell line (Cat. No. T0764) and the Applied Cell Extracellular Matrix were purchased from Applied Biological Materials Inc. (Richmond, BC, Canada).

Techniques: Gene Expression, Immunohistochemical staining, Expressing, Staining, Produced, Binding Assay, Negative Control

Journal: The FASEB Journal

Article Title: S1P 3 Receptor Mediates the Proinflammatory Effect of the Endocannabinoid 2‐Arachidonoylglycerol in Endometriotic Epithelial Cells

doi: 10.1096/fj.202502415R

Figure Lengend Snippet: 2‐AG, but not MAEA, increases the expression and release of different pro‐inflammatory factors in endometriotic epithelial cells. (A) Endometriotic epithelial cells were serum‐starved for 18 h and treated with increasing concentrations of 2‐AG and MAEA for 24 h. mRNA quantitative analysis of COX2, IL‐1β, IL‐6 and IL‐8 was performed by qPCR. Results, analyzed with the 2 −ΔΔCt method, were obtained using β‐Actin as a housekeeping gene and individual inflammatory factors of the unchallenged specimen as a reference gene. 2‐AG increases COX2, IL‐1β, IL‐6 and IL‐8 expression in a statistically significant manner (One‐way ANOVA, * p < 0.05; ** p < 0.01; **** p < 0.0001). (B) Endometriotic epithelial cells were serum‐starved for 18 h and treated with 10 μM 2‐AG or 2.5 μM MAEA for 24 h. The obtained conditioned media were screened for the content of proinflammatory markers using the Human Inflammation Array as described in Section . Results were expressed as fold increase in respect to control. 2‐AG induces the extracellular release of IL‐1β, IL‐2, IL‐6, IL‐7 and IFNγ in a statistically significant manner (Student's t ‐test * p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: Human endometriotic epithelial 12Z cell line (Cat. No. T0764) and the Applied Cell Extracellular Matrix were purchased from Applied Biological Materials Inc. (Richmond, BC, Canada).

Techniques: Expressing, Control

Journal: The FASEB Journal

Article Title: S1P 3 Receptor Mediates the Proinflammatory Effect of the Endocannabinoid 2‐Arachidonoylglycerol in Endometriotic Epithelial Cells

doi: 10.1096/fj.202502415R

Figure Lengend Snippet: 2‐AG modulates S1P signaling axis in endometriotic epithelial cells. Endometriotic epithelial cells were serum‐starved for 18 h and treated with 10 μM 2‐AG for 24 h. (A) mRNA quantitative analysis of S1P metabolic enzymes (SK1, SK2, SPP1, SPP2 and SPL) as well as molecules implicated in S1P signaling (S1P 1–5 , the specific transporter Spns2 and the SK1‐activating protein CIB1) was performed by qPCR. Results, analyzed with the 2 −ΔΔCt method, were obtained using β‐Actin as a housekeeping gene and individual targets of the unchallenged specimen as a reference gene. 2‐AG increases SK1, S1P 3 , and Spns2 mRNA levels in a statistically significant manner (Student's t ‐test * p < 0.05; ** p < 0.01). (B) Protein lysates were analyzed using SDS‐PAGE electrophoresis and WB, using specific anti‐SK1, anti‐SK2, anti‐S1P 3 , anti‐Spns2 and anti‐GAPDH antibodies. The histograms represent the densitometric analysis of 4 independent experiments. Data are the mean ± SD and are reported as band intensity normalized to the expression of GAPDH, fold change over control (set as 1). 2‐AG increases S1P 3 protein content in a statistically significant manner (Student's t ‐test * p < 0.05).

Article Snippet: Human endometriotic epithelial 12Z cell line (Cat. No. T0764) and the Applied Cell Extracellular Matrix were purchased from Applied Biological Materials Inc. (Richmond, BC, Canada).

Techniques: SDS Page, Electrophoresis, Expressing, Control

Journal: The FASEB Journal

Article Title: S1P 3 Receptor Mediates the Proinflammatory Effect of the Endocannabinoid 2‐Arachidonoylglycerol in Endometriotic Epithelial Cells

doi: 10.1096/fj.202502415R

Figure Lengend Snippet: 2‐AG pro‐inflammatory action relies on S1P 3 . (A) Serum‐starved endometriotic epithelial cells were pretreated or not with the S1P 3 antagonist TY‐52156 (10 μM) for 45 min before being challenged with 10 μM 2‐AG for 24 h. mRNA quantitative analysis of COX2, IL‐1β, IL‐6, and IL‐8 was performed by qPCR. Results, analyzed with the 2 −ΔΔCt method, were obtained using β‐Actin as a housekeeping gene and individual inflammatory factors of the unchallenged specimen as a reference gene. The blockade of S1P 3 on 2‐AG‐induced inflammatory effect (** p < 0.01; *** p < 0.001, **** p < 0.0001) was statistically significant by two‐way ANOVA followed by Bonferroni's post hoc test ( # p < 0.05; ## p < 0.01; ### p < 0.001). (B) Endometriotic epithelial cells transfected with SCR‐, S1P 1 ‐, S1P 2 ‐ and S1P 3 ‐siRNA were serum‐starved prior to being challenged with 10 μM 2‐AG for 24 h. mRNA quantitative analysis of COX2, IL‐1β, IL‐6 and IL‐8 was performed by qPCR. Results, analyzed with the 2 −ΔΔCt method, were obtained using β‐Actin as a housekeeping gene and individual inflammatory factors of the unchallenged specimen as a reference gene. The effect of S1P 3 downregulation in the reduction of 2‐AG‐induced inflammatory effect (* p < 0.05; *** p < 0.001; **** p < 0.0001) was statistically significant by two‐way ANOVA followed by Bonferroni's post hoc test ( # p < 0.05; ## p < 0.01; ### p < 0.001; #### p < 0.0001).

Article Snippet: Human endometriotic epithelial 12Z cell line (Cat. No. T0764) and the Applied Cell Extracellular Matrix were purchased from Applied Biological Materials Inc. (Richmond, BC, Canada).

Techniques: Transfection

Journal: The FASEB Journal

Article Title: S1P 3 Receptor Mediates the Proinflammatory Effect of the Endocannabinoid 2‐Arachidonoylglycerol in Endometriotic Epithelial Cells

doi: 10.1096/fj.202502415R

Figure Lengend Snippet: 2‐AG pro‐inflammatory action is SK‐independent in endometriotic epithelial cells. (A) Serum‐starved endometriotic epithelial cells were treated with 10 μM 2‐AG for different time intervals (5, 15 and 30 min). Protein lysates were analyzed using SDS‐PAGE electrophoresis and WB, using specific anti‐phospho‐SK1 (P‐SK1), anti‐phospho‐SK2 (P‐SK2) and anti‐GAPDH antibodies. The histograms represent the densitometric analysis of three independent experiments. Data are the mean ± SD and are reported as band intensity normalized to the expression of GAPDH, fold change over control (set as 1). (B) Serum‐starved endometriotic epithelial cells were treated for 30 min with 10 μM 2‐AG before being harvested and then subjected to C17‐S1P quantification by LC–MS/MS as described in the Section . Results are the mean ± SEM of three independent experiments and are reported as pmol of S1P normalized on cell number. (C) Endometriotic epithelial cells transfected with SCR‐, SK1‐ and SK2‐siRNA were serum‐starved prior to being challenged with 10 μM 2‐AG for 24 h. mRNA quantitative analysis of COX2, IL‐1β, IL‐6 and IL‐8 was performed by qPCR. Results, analyzed with the 2 −ΔΔCt method, were obtained using β‐Actin as a housekeeping gene and individual inflammatory factors of the unchallenged specimen as a reference gene. The effect of SK1 or SK2 downregulation did not significantly affect the 2‐AG inflammatory effect (* p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: Human endometriotic epithelial 12Z cell line (Cat. No. T0764) and the Applied Cell Extracellular Matrix were purchased from Applied Biological Materials Inc. (Richmond, BC, Canada).

Techniques: SDS Page, Electrophoresis, Expressing, Control, Liquid Chromatography with Mass Spectroscopy, Transfection