Activation of FGFR-related intracellular pathways in sperm exposed to FGF2.

<p>Sperm were incubated for a total 4-h period and exposed to FGF2 (0, 1, 10 and 100 ng/ml) for the last 15 min. In some aliquots, sperm were incubated for 15 min with BGJ398 (0.1 μM) before the addition of FGF2. (<b>A</b>) Immunolocalization of pERK and pAkt in sperm incubated in the absence of FGF2 (Control), with 100 ng/ml FGF2, and with BGJ398 + 100 ng/ml FGF2. Sperm were processed for immunocytochemistry, stained with anti pERK or pAkt and FITC-conjugated secondary antibodies; nuclei were stained with propidium iodide. Bar: 10 μm. (<b>B</b>) Percentage of sperm cells stained with anti pERK and anti pAkt after exposure to FGF2 in the absence (<b>left</b>) or presence of BGJ398 (<b>right</b>). Results are expressed as mean ± SEM, n = 4. * <i>P</i> < 0.05; ** <i>P</i> < 0.01 compared with Control. (<b>C</b>) Phosphorylation of ERK and Akt assessed by Western immunoblotting. Protein extracts from human sperm were subjected to SDS-PAGE and Western immunoblotting using anti pERK, ERK, pAkt and Akt antibodies. The estimated molecular weights of the protein bands are indicated on the right. (<b>D</b>) Densitometric analysis of Western immunoblotting results for pERK normalized to ERK and pAkt normalized to Akt. Results are expressed as mean ± SEM, n = 5 for ERK and n = 5 for Akt. * <i>P</i> < 0.05 and ** <i>P</i> < 0.01 compared with Control.</p

Activation of sperm FGFRs in response to FGF2.

<p>Localization of sperm pFGFRs by immunocytochemistry. Sperm were incubated for 4 h and exposed to FGF2 (0, 1, 10 and 100 ng/ml) for the last 15 min. In some aliquots, sperm were incubated for 15 min with BGJ398 (0.1 μM) before the addition of FGF2. Sperm were processed for immunocytochemistry, stained with anti pFGFR and FITC-conjugated secondary antibody; nuclei were stained with propidium iodide. (<b>A</b>) pFGFR immunolocalization in sperm incubated in the absence of FGF2 (Control), with 100 ng/ml FGF2, and with BGJ398 + 100 ng/ml FGF2. Bar: 10 μm. (<b>B</b>) Percentage of sperm cells stained with anti pFGFR antibody after exposure to different concentrations of FGF2 in the absence (<b>left</b>) or presence of BGJ398 (<b>right</b>). Results are expressed as mean ± SEM, n = 4. * <i>P</i> < 0.05; ** <i>P</i> < 0.01 compared with Control.</p

Localization of FGFRs in human sperm.

<p>Sperm cells were stained with anti FGFR1, FGFR2, FGFR3 and FGFR4 or rabbit IgG and a secondary antibody labeled with Cy3. The corresponding fields stained with FITC-PSA to assess acrosomal status are shown. Bar: 10 μm. On the right, a representative image of individual sperm is depicted; (<b>A</b>) sperm stained with anti FGFR antibody and Cy3-conjugated secondary antibody, (<b>B</b>) FITC-PSA, (<b>C</b>) merge.</p

Localization of FGFRs in human seminiferous epithelium.

<p>Immunohistochemical analysis of FGFRs in human testis using anti FGFR antibodies; rabbit IgG was included as control. The specimens were counterstained with hematoxylin. S: Sertoli cell, Sg: spermatogonia, Sc: spermatocyte, St: spermatid. Arrows indicate immunoreactivity for FGFRs in the flagellum of elongating/elongated spermatids and the arrow head indicates FGFR4 immunoreactivity in spermatid acrosome. Bar: 20 μm.</p

Expression of FGFRs in human testis and sperm.

<p>(<b>A</b>) Messenger RNA expression of testicular and sperm FGFRs assessed by RT-PCR. Messenger RNA extracted from MCF7 cells served as positive controls; negative controls without reverse transcriptase (RT Control) and without template (PCR Control) are shown. The amplicon sizes are indicated on the right. (<b>B</b>) Detection of testis and sperm FGFR protein forms using Western immunoblotting. Protein extracts from human testis and sperm were subjected to SDS-PAGE and Western immunoblotting using anti FGFR antibodies or rabbit IgG as control. The estimated molecular weights of the protein bands are indicated on the right. The experiments were performed at least 3 times obtaining similar results. Typical results are shown.</p

Effect of sperm incubation with FGF2 on sperm motility.

<p>Sperm were incubated with 0, 10 and 100 ng/ml FGF2 in the absence or in the presence of 0.1 μM BGJ398 and subjected to computer-assisted sperm analysis. (<b>A</b>) Percentages of progressive (Grade a + b) and total motility (Grade a + b + c) for aliquots incubated in the absence (<b>left</b>) or in the presence of BGJ398 (<b>right</b>). Results are expressed as mean ± SEM, n = 5. ** <i>P</i> < 0.01 compared with Control. (<b>B</b>) Individual recordings of the effect of sperm incubation with FGF2 (0, 10 and 100 ng/ml) on the percentage of total sperm motility in samples with low and high sperm motility. Each sample is identified with a different symbol (n = 12). (<b>C</b>) The percentages of sperm with Grade a, b, c and d motility in each condition (as defined in Materials and Methods) is depicted. * <i>P</i> < 0.05; ** <i>P</i> < 0.01 compared with the same Grade in Control (n = 12).</p

Data_Sheet_3_FXYD5/Dysadherin, a Biomarker of Endometrial Cancer Myometrial Invasion and Aggressiveness: Its Relationship With TGF-β1 and NF-κB Pathways.pdf

Objective: Endometrial cancer (EC) is the second most common gynecological cancer worldwide. Myometrial invasion (MI) is a key event in EC dissemination. This study aimed to evaluate FXYD5/dysadherin (FXYD5/Dys) expression in EC tissue and uterine aspirate (UA) biopsies and to assess molecular/functional changes associated with its expression in cellular models.Methods: FXYD5/Dys messenger RNA (mRNA) levels were determined in EC tissue and UA biopsies. FXYD5/Dys expression was evaluated in EC RNAseq data from The Cancer Genome Atlas (TCGA) and GENEVESTIGATOR tools. FXYD5/Dys impact on E-cadherin expression and cell behavior was assessed in EC Hec1a cells treated with transforming growth factor (TGF)-β1, stably transfected with ETV5, and transiently transfected with FXYD5/Dys small interfering RNA (siRNA) or pcDNA3-FXYD5/Dys plasmid.Results: FXYD5/Dys was associated with EC aggressiveness, finding high mRNA levels in tumors depicting MI > 50%, Grade 3, and intermediate/high risk of recurrence. FXYD5/Dys was highly expressed at the tumor invasive front compared to the superficial area. Most results were recapitulated in UA biopsies. FXYD5/Dys modulation in Hec1a cells altered cell migration/adhesion and E-cadherin expression. TGF-β1 treatment of Hec1a cells induced FXYD5/Dys expression. TCGA-UCEC RNAseq analysis revealed a positive correlation between FXYD5/Dys, TGF-β1, and plasminogen activator inhibitor (PAI)-1 mRNA levels. FXYD5/Dys induced nuclear factor (NF)-κB pathway activation in Hec1a cells. FXYD5/Dys mRNA levels positively correlated with transcriptional activation of NF-κB p65-regulated genes. Survival analysis revealed patient segregation into low- and high-risk groups, the latter depicting the highest FXYD5/Dys, PAI-1, tumor necrosis factor (TNF)-α, and TGF-β1 mRNA levels and shorter survival rates.Conclusion: FXYD5/Dys is a novel biomarker of EC progression related to TGF-β1 and NF-κB pathways that collectively promote tumor dissemination and result in poor patient prognosis.</p

Data_Sheet_2_FXYD5/Dysadherin, a Biomarker of Endometrial Cancer Myometrial Invasion and Aggressiveness: Its Relationship With TGF-β1 and NF-κB Pathways.pdf

Objective: Endometrial cancer (EC) is the second most common gynecological cancer worldwide. Myometrial invasion (MI) is a key event in EC dissemination. This study aimed to evaluate FXYD5/dysadherin (FXYD5/Dys) expression in EC tissue and uterine aspirate (UA) biopsies and to assess molecular/functional changes associated with its expression in cellular models.Methods: FXYD5/Dys messenger RNA (mRNA) levels were determined in EC tissue and UA biopsies. FXYD5/Dys expression was evaluated in EC RNAseq data from The Cancer Genome Atlas (TCGA) and GENEVESTIGATOR tools. FXYD5/Dys impact on E-cadherin expression and cell behavior was assessed in EC Hec1a cells treated with transforming growth factor (TGF)-β1, stably transfected with ETV5, and transiently transfected with FXYD5/Dys small interfering RNA (siRNA) or pcDNA3-FXYD5/Dys plasmid.Results: FXYD5/Dys was associated with EC aggressiveness, finding high mRNA levels in tumors depicting MI > 50%, Grade 3, and intermediate/high risk of recurrence. FXYD5/Dys was highly expressed at the tumor invasive front compared to the superficial area. Most results were recapitulated in UA biopsies. FXYD5/Dys modulation in Hec1a cells altered cell migration/adhesion and E-cadherin expression. TGF-β1 treatment of Hec1a cells induced FXYD5/Dys expression. TCGA-UCEC RNAseq analysis revealed a positive correlation between FXYD5/Dys, TGF-β1, and plasminogen activator inhibitor (PAI)-1 mRNA levels. FXYD5/Dys induced nuclear factor (NF)-κB pathway activation in Hec1a cells. FXYD5/Dys mRNA levels positively correlated with transcriptional activation of NF-κB p65-regulated genes. Survival analysis revealed patient segregation into low- and high-risk groups, the latter depicting the highest FXYD5/Dys, PAI-1, tumor necrosis factor (TNF)-α, and TGF-β1 mRNA levels and shorter survival rates.Conclusion: FXYD5/Dys is a novel biomarker of EC progression related to TGF-β1 and NF-κB pathways that collectively promote tumor dissemination and result in poor patient prognosis.</p

Data_Sheet_1_FXYD5/Dysadherin, a Biomarker of Endometrial Cancer Myometrial Invasion and Aggressiveness: Its Relationship With TGF-β1 and NF-κB Pathways.zip

Objective: Endometrial cancer (EC) is the second most common gynecological cancer worldwide. Myometrial invasion (MI) is a key event in EC dissemination. This study aimed to evaluate FXYD5/dysadherin (FXYD5/Dys) expression in EC tissue and uterine aspirate (UA) biopsies and to assess molecular/functional changes associated with its expression in cellular models.Methods: FXYD5/Dys messenger RNA (mRNA) levels were determined in EC tissue and UA biopsies. FXYD5/Dys expression was evaluated in EC RNAseq data from The Cancer Genome Atlas (TCGA) and GENEVESTIGATOR tools. FXYD5/Dys impact on E-cadherin expression and cell behavior was assessed in EC Hec1a cells treated with transforming growth factor (TGF)-β1, stably transfected with ETV5, and transiently transfected with FXYD5/Dys small interfering RNA (siRNA) or pcDNA3-FXYD5/Dys plasmid.Results: FXYD5/Dys was associated with EC aggressiveness, finding high mRNA levels in tumors depicting MI > 50%, Grade 3, and intermediate/high risk of recurrence. FXYD5/Dys was highly expressed at the tumor invasive front compared to the superficial area. Most results were recapitulated in UA biopsies. FXYD5/Dys modulation in Hec1a cells altered cell migration/adhesion and E-cadherin expression. TGF-β1 treatment of Hec1a cells induced FXYD5/Dys expression. TCGA-UCEC RNAseq analysis revealed a positive correlation between FXYD5/Dys, TGF-β1, and plasminogen activator inhibitor (PAI)-1 mRNA levels. FXYD5/Dys induced nuclear factor (NF)-κB pathway activation in Hec1a cells. FXYD5/Dys mRNA levels positively correlated with transcriptional activation of NF-κB p65-regulated genes. Survival analysis revealed patient segregation into low- and high-risk groups, the latter depicting the highest FXYD5/Dys, PAI-1, tumor necrosis factor (TNF)-α, and TGF-β1 mRNA levels and shorter survival rates.Conclusion: FXYD5/Dys is a novel biomarker of EC progression related to TGF-β1 and NF-κB pathways that collectively promote tumor dissemination and result in poor patient prognosis.</p

Expression analyses of E-cadherin and EMT-related markers in OC cell lines grown under anchorage-independent conditions.

<p><b>Assessment of their aggregation and survival capacities. (A)</b> Phase contrast images of 48 hour-aggregates (100x and 200x magnifications). <b>(B)</b> Plot of the area (px²: pixeles²) and number (black spots) of 48 hour-aggregates in 4 drops of each cell line. <b>(C)</b> Western immunoblotting analyses of E-cadherin, N-cadherin, pan-cytokeratin and vimentin in 48 hour-aggregates. β-tubulin served as total protein loading control. <b>(D)</b> Quantitative real time PCR analyses of E-cadherin and N-cadherin mRNA expression levels in 48 hour-aggregates. <b>(E)</b> Fluorescent immunocytochemistry analysis of E-cadherin and N-cadherin in 48 hour-aggregates (400x magnification). A merge image of both cadherins is also included. <b>(F)</b> Cell death assessed by means of PI staining in 48 hour-aggregates. Images were taken using an inverted microscope with phase contrast and red fluorescence after PI staining of disaggregated cells (100x magnification) (top). Cell death (%) was plotted (bottom) (***p<0.001). <b>(G)</b> Western immunoblotting analysis of PARP-1 on 48 hour-aggregates (left). Relative expression (%) of cleaved versus FL PARP-1 form (right).</p