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: Placenta

Article Title: Krüppel-like factor 6 (KLF6) requires its amino terminal domain to promote villous trophoblast cell fusion.

doi: 10.1016/j.placenta.2021.12.006

Figure Lengend Snippet: Fig. 4. KLF6 decreases BeWo cell proliferation and promotes their differentiation in a p21-dependent manner. A- Percentage of BrdU positive cells in each condition expressed as the mean ± SEM (*p < 0.05, t-test, n = 2). For each condition, at least 1000 cells from randomly selected fields were counted. B- Repre sentative western blot images for p21 and α-tubulin in KLF6-B and EV-B cells. Bar graphs represent p21 expression level in KLF6-B relative to EV-B defined as 1. Values are expressed as the mean ± SEM (*p < 0.05, one sample t-test, n = 3). C- Western blot analysis of p21 and KLF6 proteins in KLF6-B cells transfected with a specific siRNA for p21 (sip21) or with the scramble sequence (SCR), α-tubulin was included as loading control. D- Fusion index of KLF6-B cells transfected with sip21, SCR, or non-transfected cells expressed relative to the fusion induced in KLF6-B defined as 1. The mean ± SEM is shown (*p < 0.05, ANOVA, post-test Tukey’s multiple comparison, ns: not significant, n = 3). E- Representative images of desmoplakin immunodetection and nuclei staining with Hoechst of KLF6-B cells transfected with SCR or sip21. White dotted lines demarcate syncytial-like structures. Scale bar = 100 μm. The insets show a magnified image of the boxed regions.

Article Snippet: Antibodies used were: mouse monoclonal anti-KLF6 (1:1000; 2c11) whose specificity was previously determined [25], mouse monoclonal anti-α-tubulin (1:3000; T9026, Sigma-Aldrich), rabbit polyclonal anti-KLF6 (1:50; R-173, Santa Cruz Biotechnology), rabbit polyclonal anti-syncytin-1 (1:500; z-25, Santa Cruz Biotechnology), rabbit monoclonal anti-p21 (1:1000, Cell Signaling), rabbit polyclonal anti-β-hCG (1:500; A0231, Dako), rabbit polyclonal anti-GRP78 (1:1000, Cell Signaling) and mouse monoclonal anti-β-actin (1:1000; A2228, Sigma-Aldrich).

Techniques: Western Blot, Expressing, Transfection, Sequencing, Control, Comparison, Immunodetection, Staining