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