Effect of steroid hormones (E₂ and P₄) on the viability of leiomyoma and myometrial adjacent cells.

<p>(A) Leiomyoma cells (5 × 10³) and myometrial adjacent cells were treated with E₂ (100 nmol/L) and P4 (100 nmol/L) for 24 h in 96-well microtiter plates. Cell viability was assessed by the MTT reduction test. (B) Phase contrast–confluent culture of leiomyoma and myometrial adjacent cells after treat with E₂ (100 nmol/L) and P₄ (100 nmol/L). The statistical significance was evaluated using one-way ANOVA followed by the Tukey's test. A p-value of ≤0.05 was considered to indicate significance (*). These experiments were performed with cultured primary cells from specimens collected from patients.</p

Cell viability by the MTT assay.

<p>(A and B) Leiomyoma and myometrial adjacent cells (1 × 10⁴) were maintained in serum deprivation with different concentrations of FBS (0%-10%) for 24 and 48 h in 96-well microtiter plates. (C) Cell viability of uterine leiomyoma cells and myometrial adjacent cells cultured in two concentrations of FBS (2% and 10%); cells (5 × 10³) were seed on plastic and on a collagen type I coated plates. (D and E) Phase contrast of confluent culture of leiomyoma cells and myometrial adjacent cells on collagen type I coated plates. The morphology of leiomyoma cells is not altered; these cells were less spread than the myometrial adjacent cells when seeded onto collagen type I-coated plates. Mycoplasma contamination was not observed in any of the processed tissues.</p

Characteristics of Samples Used in the Study.

<p>Characteristics of Samples Used in the Study.</p

Detection of signaling phosphoproteins by Immunoblot analysis in leiomyoma and myometrial adjacent cells.

<p>Leiomyoma and myometrial adjacent cells (5 x 10⁵) were treated with E₂ (100 nmol/L) and P₄ (100 nmol/L) for 16 h in 6-well microtiter plates; lysate proteins were separated by 10% SDS-PAGE and electro-transferred to nitrocellulose membranes. Membranes were blocked and incubated with rabbit primary antibodies, (A and C) anti-phospho-Src (Tyr-416), anti-Src, anti-phospho-FAK (Tyr-397), anti-FAK, anti- phospho-Erk1/2 MAPK, anti-Erk1/2 MAPK, (E) anti-p130Cas Y165, (F) anti-p130Cas Y410, (H and J) anti-phospho-Akt, anti-Akt, and anti-β-actin. (B, D, G, I, and L) Graph bars represent the densitometric analyses of the immunoblotting results. The results are represented as band intensities in arbitrary units relative to the respective total phopho-proteins load and total control (β-actin) load. Antibody binding was visualized by chemiluminescence, and the relative levels of these proteins were determined by the densitometric analyses. These experiments were performed with cultured primary cells from specimens collected from patients (*p<0.01).</p

Immunophenotype of leiomyoma and myometrial cells from women with myoma uterine.

<p>Adhered and spread cells after tissue disaggregation in collagenase following with primary explants. Cells are shown under phase contrast microscopy and indirect immunofluorescence for phalloidin, fibronectin-integrin β1/CD29, vimentin, and DAPI (blue, for nuclei). (A-B) Phase contrast in the confluent culture of leiomyoma cells after three days. (A) Low density (B) high density (magnification, ×400). Mycoplasma contamination was not observed in any of the processed tissues. (C) Log-phase growth rate by cell counting–growth characteristics of leiomyoma cells, myometrial adjacent cells, and co-cultured myometrial adjacent (as feeders) with leiomyoma cells (on plastic surface). (D-F) Analysis of myometrial markers by confocal microscopy; vimentin and fibronectin integrin β1/CD29. (D) Cytoskeletal organization (Phalloidin, Alexa-594-red); (E) integrin β1/CD29 (FITC-488, green); and (F) Co-localization integrin β1(FITC-488, green) and vimentin (Alexa-594-red). Bar, 10 μm.</p