Journal: bioRxiv

Article Title: Mechanical Cues Regulate Estrogen and Progesterone-Induced Nascent ECM Deposition by Human Endometrial Stromal Cells

doi: 10.1101/2025.10.14.682403

Figure Lengend Snippet: a Schematic illustrating the female reproductive system including the uterus with the endometrium and myometrium, the ovary and fallopian tubes. Inset shows endometrial stromal cells embedded in extracellular matrix (ECM) containing a variety of proteins (e.g., laminin, collagens and fibronectin). b Schematic illustrating the relative levels of estrogen and progesterone during the menstrual cycle. c Schematic illustrating the endometrial tissue during the proliferative and remodeling phase of the menstrual cycle.

Article Snippet: Primary endometrial stromal cells (PCS-460-010, ATCC; 25-year-old donor) were cultured in fibroblast growth media, containing 10% fetal bovine serum (PCS-201-030 and PCS-201-041, ATCC), and passaged twice prior cryopreservation.

Techniques:

Journal: bioRxiv

Article Title: Mechanical Cues Regulate Estrogen and Progesterone-Induced Nascent ECM Deposition by Human Endometrial Stromal Cells

doi: 10.1101/2025.10.14.682403

Figure Lengend Snippet: a Schematic illustrating the structure of progesterone, estrogen, and 3’,5’-cyclic adenosine monophosphate (cAMP). b Timeline of endometrial stromal cell seeding on glass (-24 hours) and culture for 24 hours to reach confluency before an additional culture up to 96 hours without hormones (Ctrl) or with hormones/cAMP (+Hrm). Cells were fixed at 0, 24, 48, 72, and 96 hours for image analysis. c Schematic illustrating the change in endometrial stromal cell morphology in response to hormonal treatment leading to the secretion of hormones, such as prolactin. d Representative fluorescent images of Phalloidin (F-actin) and Hoechst (nucleus) of endometrial stromal cells cultured for 0, 24, and 48 hours without hormones (Ctrl, scale bars = 100 µm, insets = 55 µm). e Quantification of size and number of nuclei of endometrial stromal cells cultured for 0, 24, and 48 hours in control media (n = 3 replicates, 5 images per replicate from one representative experiment, **p≤0.001, **** p≤0.0001, ns = not significant by one-way ANOVA). f Representative fluorescent images of Phalloidin (F-actin) and Hoechst (nucleus) of endometrial stromal cells cultured for 0, 24, and 48 hours with hormones (+Hrm, scale bars = 100µm, inset = 55µm). g Quantification of size and number of nuclei of endometrial stromal cells cultured for 0, 24, and 48 hours in hormone media (n = 3 replicates, 5 images per replicate from one representative experiment, **p≤0.001, **** p≤0.0001, ns = not significant by one-way ANOVA).

Article Snippet: Primary endometrial stromal cells (PCS-460-010, ATCC; 25-year-old donor) were cultured in fibroblast growth media, containing 10% fetal bovine serum (PCS-201-030 and PCS-201-041, ATCC), and passaged twice prior cryopreservation.

Techniques: Cell Culture, Control

Journal: bioRxiv

Article Title: Mechanical Cues Regulate Estrogen and Progesterone-Induced Nascent ECM Deposition by Human Endometrial Stromal Cells

doi: 10.1101/2025.10.14.682403

Figure Lengend Snippet: a Timeline of endometrial stromal cell seeding: culture for 24 hours to attach to the substrate before additional culture for 48 hours without hormones (Ctrl) or with hormones/cAMP (+Hrm) prior harvesting for image analysis. b Schematic illustrating norbornene-modified hyaluronic acid (NorHA) hydrogels with different elastic moduli using 1.29 (low) or 3.24 mM (high) dithiol. c Quantification of compressive Young’s modulus of 1.29 mM (low) and 3.24 mM (high) DTT crosslinked NorHA hydrogels (n = 3 hydrogels, ***p=0.0001 by Student’s t -test). d Representative fluorescent images of endometrial stromal cells cultured for 48 hours atop 5 kPa and 15 kPa hydrogels without hormones (Ctrl, scale bars = 100µm, inset = 55µm). e Quantification of number of nuclei and area of nascent ECM deposition, normalized to number of nuclei, of endometrial stromal cells cultured for 48 hours atop 5 kP and 15 kPa hydrogels (5kPa: n = 48 images from 4 independent experiments, 15 kPa: n = 40 images from 3 independent experiments, *p≤0.05, ns = not significant by Students t -test). f Representative fluorescent images and quantification of number of nuclei and area of nascent ECM, normalized to number of nuclei, deposited by endometrial stromal cells cultured for 48 hours atop 5 kPa hydrogels with hormones (+Hrm, scale bars = 100 µm, insets = 55 µm, n = 48 images from 4 independent experiments, ****p≤0.0001 by Students t -test). g Representative fluorescent images and quantification of number of nuclei and area of nascent ECM, normalized to number of nuclei, deposited by endometrial stromal cells cultured for 48 hours atop 15 kPa hydrogels with hormones (+Hrm, scale bars = 100 µm, insets = 55 µm, n = 38 images from 3 independent experiments, *p ≤ 0.05 by Students t -test).

Article Snippet: Primary endometrial stromal cells (PCS-460-010, ATCC; 25-year-old donor) were cultured in fibroblast growth media, containing 10% fetal bovine serum (PCS-201-030 and PCS-201-041, ATCC), and passaged twice prior cryopreservation.

Techniques: Modification, Cell Culture

Journal: bioRxiv

Article Title: Mechanical Cues Regulate Estrogen and Progesterone-Induced Nascent ECM Deposition by Human Endometrial Stromal Cells

doi: 10.1101/2025.10.14.682403

Figure Lengend Snippet: a Representative fluorescent images of cells seeded on 5 kPa gels without (Ctrl) and with hormones (+Hrm), stained with Hoechst (nuclei), Phalloidin (F-actin), DBCO-488 (nascent ECM), and anti-Fibronectin antibody (scale bars = 100 µm, insets = 55 µm). b Quantification of area of fibronectin deposited normalized to number of endometrial stromal cells cultured on 5 kPa hydrogels for 48 hours with and without hormones (5 kPa gels: n = 28 images from 3 independent experiments, ****p≤0.0001 by Student’s t -test). c Representative fluorescent images of cells seeded on 15 kPa gels without and with hormones, stained with Hoechst, Phalloidin, DBCO-488, and anti-Fibronectin antibody (scale bars = 100 µm, insets = 55 µm). d Quantification of area of fibronectin deposited normalized to number of endometrial stromal cells cultured on 15 kPa hydrogels for 48 hours with and without hormones (15 kPa gels: n = 19 images from 2 independent experiments, **p ≤ 0.01 by Student’s t -test). e Representative fluorescent images of cells seeded on glass coverslips without and with hormones, stained with Hoechst, Phalloidin, DBCO-488, and anti-Fibronectin antibody (scale bars = 100 µm, insets = 55 µm). f Quantification of area of fibronectin deposited normalized to number of endometrial stromal cells cultured on glass coverslips for 48 hours with and without hormones (glass: n = 15 images from one representative experiment, ns = not significant by Student’s t -test).

Article Snippet: Primary endometrial stromal cells (PCS-460-010, ATCC; 25-year-old donor) were cultured in fibroblast growth media, containing 10% fetal bovine serum (PCS-201-030 and PCS-201-041, ATCC), and passaged twice prior cryopreservation.

Techniques: Staining, Cell Culture