Journal: PLoS ONE

Article Title: A FOXM1 Dependent Mesenchymal-Epithelial Transition in Retinal Pigment Epithelium Cells

doi: 10.1371/journal.pone.0130379

Figure Lengend Snippet: A. Quantification of change in cell density (number of DAPI positive nuclei per cm 2 imaged area) upon FOXM1 overexpression or knockdown, 72h post transfection. Data is normalized to appropriate controls (Empty vector for pFOXM1 and non-targeting siRNA for siFOXM1). Bars represent Mean + SD (n = 4). P<0.0001 (Student’s t-test). B. Heatmap showing changes in gene expression of a panel of representative markers over a timecourse of RPE culture where cells are seeded at high (100000 cells/cm 2 ) or low (8000 cells/cm 2 ) density. C. Plot showing differential expression of BMP7 and Wnt5B transcripts extrapolated from the microarray data. The shaded area represents 95% confidence intervals around the point estimates (circles) of the difference between the mean high density expression vs the mean low density expression.

Article Snippet: Where required, media was supplemented with recombinant human Wnt5B (500ng/ml; R&D Systems), BMP-4/7 (75ng/ml; R&D Systems), Thiostrepton (Sigma), LDN-193189 (10μM; Stemgent), WAY-262611 (10μM; Enzo Lifesciences).

Techniques: Over Expression, Knockdown, Transfection, Plasmid Preparation, Gene Expression, Quantitative Proteomics, Microarray, Expressing

Journal: PLoS ONE

Article Title: A FOXM1 Dependent Mesenchymal-Epithelial Transition in Retinal Pigment Epithelium Cells

doi: 10.1371/journal.pone.0130379

Figure Lengend Snippet: A. Immunocytochemistry for PMEL17 where cells are seeded at either low density (16000 cells/cm 2 ) in the presence or absence of BMP4/7 (top left) or at high density (25000 cells/cm 2 ) in the presence or absence of Wnt5B (bottom left) and cultured for a period of 14 days. Also shown is the expression of BEST1 under the same conditions (top and bottom right). ACTB and B2M are used as housekeeping genes. Bars represent Mean + SD (n = 3). B. Quantification of immunocytochemistry for % CRALBP at Day 21 where cells are either treated with media alone (Control) or media supplemented with 10μM LDN-193189 added at Day 2,4,6,8,11,14 or 18. * indicates significant difference between control and compound treatment (One way ANOVA with Dunnett’s multiple comparisons). C. Quantification of immunocytochemistry for % CRALBP at Day 28 where cells are either treated with media alone (Control) or media supplemented with 10μM WAY-262611 added at Day 2,7,14 or 21. * indicates significant difference between control and compound treatment (One way ANOVA with Dunnett’s multiple comparisons). D. qPCR based measurement of BMP7 and Wnt5B transcript expression at Day 10 post siFOXM1 transfection (relative to transfection with non-targeting siRNA used as a control). GAPDH , HPRT1 and IPO8 were used as housekeeping genes. Bars represent Mean + SD (n = 3). P<0.05 (Student’s t-test).

Article Snippet: Where required, media was supplemented with recombinant human Wnt5B (500ng/ml; R&D Systems), BMP-4/7 (75ng/ml; R&D Systems), Thiostrepton (Sigma), LDN-193189 (10μM; Stemgent), WAY-262611 (10μM; Enzo Lifesciences).

Techniques: Immunocytochemistry, Cell Culture, Expressing, Control, Transfection

Journal: PLoS ONE

Article Title: A FOXM1 Dependent Mesenchymal-Epithelial Transition in Retinal Pigment Epithelium Cells

doi: 10.1371/journal.pone.0130379

Figure Lengend Snippet: RPE first acquire a mesenchymal morphology upon dissociation and culture followed by proliferation and mesenchymal-epithelial transition to re-uptake an epithelial phenotype. Proliferation of RPE is directly regulated by FOXM1 which also affects expression of BMP7 and Wnt5B by an unknown mechanism. Both these activities are required for successful MET and epithelialization.

Article Snippet: Where required, media was supplemented with recombinant human Wnt5B (500ng/ml; R&D Systems), BMP-4/7 (75ng/ml; R&D Systems), Thiostrepton (Sigma), LDN-193189 (10μM; Stemgent), WAY-262611 (10μM; Enzo Lifesciences).

Techniques: Expressing

Journal: Frontiers in Endocrinology

Article Title: The Effect of Wnt Pathway Modulators on Human iPSC-Derived Pancreatic Beta Cell Maturation

doi: 10.3389/fendo.2019.00293

Figure Lengend Snippet: Experimental design. (A) With this experimental set-up we aimed to assess whether Wnt-modulation could drive maturation of S7 cells toward a phenotype that closer resembles that of β-cells as found in adult human islets. To assess the effects of Wnt-modulation of S7 cells, the Wnt-modulated cells were compared to un-stimulated S7 cells as well as to adult human islets. (B) S7d7 cell cultures were treated for 4 h with either WNT3A (light green), WNT4 (green), WNT5A (orange), WNT5B (red), a combination of WNT5A&5B (dark red) for stimulation of the canonical or non-canonical Wnt pathways, or TKi (yellow) to block endogenous Wnt signaling in S7d7 cell cultures. The Wnt-modulated S7 cells were maintained in differentiation culture for 48 h prior to harvest for downstream analysis [including proteomics analysis and immunofluorescence (IF)].

Article Snippet: S7 cells were in parallel stimulated with Wnt5A (645-WN, R&D Systems) (400 ng/mL), Wnt5B (7347-WN, R&D Systems) (400 ng/mL), a combination of Wnt5A/Wnt5B (400 ng/mL/80 ng/mL), Wnt4 (6076-WN, R&D Systems) (100 ng/mL), Wnt3A (5036-WN, R&D Systems) (200 ng/mL), and the tankyrase inhibitor G007-LK (5 μmol/L), for 4 h and kept 48 h in S7 differentiation media before harvest.

Techniques: Blocking Assay, Immunofluorescence

Journal: Frontiers in Endocrinology

Article Title: The Effect of Wnt Pathway Modulators on Human iPSC-Derived Pancreatic Beta Cell Maturation

doi: 10.3389/fendo.2019.00293

Figure Lengend Snippet: S7-cells show heterogeneous expression of downstream targets 48 h after Wnt-modulator treatment. (A) Schematic overview of key proteins of the canonical and non-canonical Wnt signaling pathways, in brief, Wnt ligand binds to its receptor Frizzled and co-receptors LRP5/6, receptor tyrosine kinase, (or ROR2) and transmits the signal via disheveled (Dvl) into the cytoplasm to activate the canonical Wnt pathway, or functions through non-canonical planar cell polarity (PCP) and Wnt/Ca 2+ . In the canonical Wnt signaling pathway, β-catenin accumulates in the cytoplasm and translocate to the nucleus to act as a transcription coactivator for the TCF/LEF transcription factor family. Without Wnt, β-catenin is degraded by the destruction complex, composed of Axin, adenomatosis polyposis coli (APC), glycogen synthase kinase 3β (GSK3β) and casein kinase 1α (CK1α). The non-canonical Wnt/PCP pathway is thought to use Ryk or ROR2 for activation; Dvl is recruited to form a complex with disheveled-associated activator of morphogenesis 1 (DAAM1). DAAM1 activates Rho that again activates Rho-associated kinase (ROCK). Dvl can also form a complex with Rac1 to activate JNK via the MAPK pathway. In the Wnt/Ca 2+ pathway, binding of Wnt to Frizzled activates a trimetic G-protein leading to activation of PLC to cleave PIP2 to form DAG and IP3. IP3 binds to its receptor on the endoplasmic reticulum and calcium is released. Increased concentrations of calcium and DAG can again activate PKC and CaMKII. (B) A selection of interaction partners of the selected Wnt-ligands ( – ). (C) IF of β-catenin, BMP4, ROR2 and c-JUN in S7 cells, WNT3A, WNT4, WNT5A, WNT5B treated S7 cells, respectively. Scale bar 50 μm.

Article Snippet: S7 cells were in parallel stimulated with Wnt5A (645-WN, R&D Systems) (400 ng/mL), Wnt5B (7347-WN, R&D Systems) (400 ng/mL), a combination of Wnt5A/Wnt5B (400 ng/mL/80 ng/mL), Wnt4 (6076-WN, R&D Systems) (100 ng/mL), Wnt3A (5036-WN, R&D Systems) (200 ng/mL), and the tankyrase inhibitor G007-LK (5 μmol/L), for 4 h and kept 48 h in S7 differentiation media before harvest.

Techniques: Expressing, Protein-Protein interactions, Activation Assay, Binding Assay, Selection

Journal: Oncotarget

Article Title: Aberrant expression of homeobox gene SIX1 in Hodgkin lymphoma.

doi: 10.18632/oncotarget.5556

Figure Lengend Snippet: Figure 4: WNT-signalling activates SIX1 expression in HL. A. RQ-PCR analysis of L-428 and U-HO1 cells after treatment with FGF2, TGFb and WNT5B demonstrates activation of SIX1 expression by the WNT-pathway. B. RQ-PCR analysis of L-428 and U-HO1 cells after treatment with WNT-signalling inhibitor IWR confirms activation of SIX1 expression by WNT-signalling.

Article Snippet: Treatments of cell lines were performed for 20 h with 10 μg/ml Trichostatin A (TSA) (Sigma, Taufkirchen, Germany), 10 μM XMD8–92 (Biozol, Eching, Germany), 10 μM IWR1 (R&D Systems, Wiesbaden, Germany), 20 ng/ml recombinant human Bone Morphogenic Protein 4 (BMP4) protein, 20 ng/ml Fibroblast Growth Factor 2 (FGF2) protein, 20 ng/ml Transforming Growth Factor beta (TGFb), or 20 ng/ml WNT5B protein (R&D Systems).

Techniques: Expressing, Activation Assay