Journal: PLoS ONE

Article Title: Krüppel-Like Factor 6 Expression Changes during Trophoblast Syncytialization and Transactivates ßhCG and PSG Placental Genes

doi: 10.1371/journal.pone.0022438

Figure Lengend Snippet: A- Isolated mononuclear villous CTB cells cultured during the indicated hours and stained for KLF6 immunofluorescence detection (middle panels) with the polyclonal R-173 (green) anti-KLF6 antibody. Nuclei were counterstained with Hoechst 33342 dye (blue) and the overlay is shown (right panels). B- Confocal microscopy imaging of KLF6 at the indicated time points of the differentiation process. KLF6 was labelled with the polyclonal R-173 antibody (left panels) and DNA was stained with propidium iodide (IP) (middle panel). Overlay is shown in the right panels. C- Fluorescence intensity profile of KLF6 (green) and IP (red) along the yellow line shown in the confocal microscopy images. D- Morphological and biochemical differentiation of isolated mononuclear CTB cells were confirmed by the disappearance of desmoplakin intercellular staining (red), the appearance of multinucleated structures and the expression of PSG proteins (green). Original magnification, x1000. Scale bar, 10 µm. Immunofluorescence assays were performed with at least three different CTB purifications and representative figures are shown.

Article Snippet: Cells were then rinsed with PBS three times, blocked with 2.5% normal goat serum in 0.2% Tween-20 in PBS (PBST) and with 0.5% fish skin gelatin in PBST, and then incubated with the following primary antibodies: rabbit polyclonal anti-KLF6 (anti-Zf9, R-173, Santa Cruz Biotech; 1∶50), mouse monoclonal anti-KLF6 (clone 2c11, 1∶150) whose specificity was previously determined , mouse anti-desmosomal protein (0.045 mg/mL, ZK-31, Sigma Chemical Co.; 1∶400), rabbit polyclonal anti-human chorionic gonadotropin (hCG, A0231, Dako; 1∶500), mouse monoclonal anti-cytokeratin 7 (Dako, Clone OV-TL 12/30) and rabbit polyclonal anti-PSG (A0131, Dako; 1∶100).

Techniques: Isolation, Cell Culture, Staining, Immunofluorescence, Confocal Microscopy, Imaging, Fluorescence, Expressing

Journal: PLoS ONE

Article Title: Krüppel-Like Factor 6 Expression Changes during Trophoblast Syncytialization and Transactivates ßhCG and PSG Placental Genes

doi: 10.1371/journal.pone.0022438

Figure Lengend Snippet: A- JEG-3 cells were cultured in the presence of 1 µM methotrexate to induce cell differentiation during the indicted times and KLF6 mRNA expression was quantified by qRT-PCR (ABI 7500, Applied Biosystems). B- JEG-3 cells transfected with the empty (white bars) or the KLF6 expression vector (black bars) were harvested 24 h after transfection and PSG , βhCG and GCM1 gene expression was quantified by qRT- PCR (ABI 7500, Applied Biosystems). For A and B, results were normalized to cyclophilin A and expressed according to the 2 −ΔΔCt method using as calibrator the mRNA level obtained from the control condition. Data are presented as mean ±SEM of three independent experiments performed in triplicates and a one-sample t-test was used to determine whether experimental values were significantly different from the control value set as 1 (*p<0.05). C- Western blot detection of KLF6, PSG, βhCG and GCM1 in protein extracts of JEG-3 cells transfected with the empty (left lane) or KLF6 expression vector (right lane). α-tubulin was used as a loading control in each assay. Representative western blots are shown and the bar graph shows the densitometric analysis of three different experiments. (*p<0.05) D - JEG-3 cells were transiently transfected with the KLF6 expression vector and 24 h later they were immunostained for the detection of KLF6 (red) and βhCG (green) with a monoclonal anti-KLF6 and a polyclonal anti-βhCG antibodies, respectively. Nuclei were counterstained with Hoechst 33342 dye (blue), and the merge of the three channels is shown on the right side. Bar = 10 µm. Original magnification: ×1000. Representative images from three independent transfections are shown. Arrowheads, JEG-3 cells overexpressing KLF6; arrows, cells positive for βhCG.

Article Snippet: Cells were then rinsed with PBS three times, blocked with 2.5% normal goat serum in 0.2% Tween-20 in PBS (PBST) and with 0.5% fish skin gelatin in PBST, and then incubated with the following primary antibodies: rabbit polyclonal anti-KLF6 (anti-Zf9, R-173, Santa Cruz Biotech; 1∶50), mouse monoclonal anti-KLF6 (clone 2c11, 1∶150) whose specificity was previously determined , mouse anti-desmosomal protein (0.045 mg/mL, ZK-31, Sigma Chemical Co.; 1∶400), rabbit polyclonal anti-human chorionic gonadotropin (hCG, A0231, Dako; 1∶500), mouse monoclonal anti-cytokeratin 7 (Dako, Clone OV-TL 12/30) and rabbit polyclonal anti-PSG (A0131, Dako; 1∶100).

Techniques: Cell Culture, Cell Differentiation, Expressing, Quantitative RT-PCR, Transfection, Plasmid Preparation, Western Blot

Journal: PLoS ONE

Article Title: Krüppel-Like Factor 6 Expression Changes during Trophoblast Syncytialization and Transactivates ßhCG and PSG Placental Genes

doi: 10.1371/journal.pone.0022438

Figure Lengend Snippet: A- Isolated mononuclear villous CTB cells cultured during the indicated hours and stained for KLF6 immunofluorescence detection (middle panels) with the polyclonal R-173 (green) anti-KLF6 antibody. Nuclei were counterstained with Hoechst 33342 dye (blue) and the overlay is shown (right panels). B- Confocal microscopy imaging of KLF6 at the indicated time points of the differentiation process. KLF6 was labelled with the polyclonal R-173 antibody (left panels) and DNA was stained with propidium iodide (IP) (middle panel). Overlay is shown in the right panels. C- Fluorescence intensity profile of KLF6 (green) and IP (red) along the yellow line shown in the confocal microscopy images. D- Morphological and biochemical differentiation of isolated mononuclear CTB cells were confirmed by the disappearance of desmoplakin intercellular staining (red), the appearance of multinucleated structures and the expression of PSG proteins (green). Original magnification, x1000. Scale bar, 10 µm. Immunofluorescence assays were performed with at least three different CTB purifications and representative figures are shown.

Article Snippet: Cells were then rinsed with PBS three times, blocked with 2.5% normal goat serum in 0.2% Tween-20 in PBS (PBST) and with 0.5% fish skin gelatin in PBST, and then incubated with the following primary antibodies: rabbit polyclonal anti-KLF6 (anti-Zf9, R-173, Santa Cruz Biotech; 1∶50), mouse monoclonal anti-KLF6 (clone 2c11, 1∶150) whose specificity was previously determined , mouse anti-desmosomal protein (0.045 mg/mL, ZK-31, Sigma Chemical Co.; 1∶400), rabbit polyclonal anti-human chorionic gonadotropin (hCG, A0231, Dako; 1∶500), mouse monoclonal anti-cytokeratin 7 (Dako, Clone OV-TL 12/30) and rabbit polyclonal anti-PSG (A0131, Dako; 1∶100).

Techniques: Isolation, Cell Culture, Staining, Immunofluorescence, Confocal Microscopy, Imaging, Fluorescence, Expressing

Journal: PLoS ONE

Article Title: Krüppel-Like Factor 6 Expression Changes during Trophoblast Syncytialization and Transactivates ßhCG and PSG Placental Genes

doi: 10.1371/journal.pone.0022438

Figure Lengend Snippet: A- JEG-3 cells were cultured in the presence of 1 µM methotrexate to induce cell differentiation during the indicted times and KLF6 mRNA expression was quantified by qRT-PCR (ABI 7500, Applied Biosystems). B- JEG-3 cells transfected with the empty (white bars) or the KLF6 expression vector (black bars) were harvested 24 h after transfection and PSG , βhCG and GCM1 gene expression was quantified by qRT- PCR (ABI 7500, Applied Biosystems). For A and B, results were normalized to cyclophilin A and expressed according to the 2 −ΔΔCt method using as calibrator the mRNA level obtained from the control condition. Data are presented as mean ±SEM of three independent experiments performed in triplicates and a one-sample t-test was used to determine whether experimental values were significantly different from the control value set as 1 (*p<0.05). C- Western blot detection of KLF6, PSG, βhCG and GCM1 in protein extracts of JEG-3 cells transfected with the empty (left lane) or KLF6 expression vector (right lane). α-tubulin was used as a loading control in each assay. Representative western blots are shown and the bar graph shows the densitometric analysis of three different experiments. (*p<0.05) D - JEG-3 cells were transiently transfected with the KLF6 expression vector and 24 h later they were immunostained for the detection of KLF6 (red) and βhCG (green) with a monoclonal anti-KLF6 and a polyclonal anti-βhCG antibodies, respectively. Nuclei were counterstained with Hoechst 33342 dye (blue), and the merge of the three channels is shown on the right side. Bar = 10 µm. Original magnification: ×1000. Representative images from three independent transfections are shown. Arrowheads, JEG-3 cells overexpressing KLF6; arrows, cells positive for βhCG.

Article Snippet: Cells were then rinsed with PBS three times, blocked with 2.5% normal goat serum in 0.2% Tween-20 in PBS (PBST) and with 0.5% fish skin gelatin in PBST, and then incubated with the following primary antibodies: rabbit polyclonal anti-KLF6 (anti-Zf9, R-173, Santa Cruz Biotech; 1∶50), mouse monoclonal anti-KLF6 (clone 2c11, 1∶150) whose specificity was previously determined , mouse anti-desmosomal protein (0.045 mg/mL, ZK-31, Sigma Chemical Co.; 1∶400), rabbit polyclonal anti-human chorionic gonadotropin (hCG, A0231, Dako; 1∶500), mouse monoclonal anti-cytokeratin 7 (Dako, Clone OV-TL 12/30) and rabbit polyclonal anti-PSG (A0131, Dako; 1∶100).

Techniques: Cell Culture, Cell Differentiation, Expressing, Quantitative RT-PCR, Transfection, Plasmid Preparation, Western Blot