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: Nature Communications

Article Title: Spatiotemporal interplay between epithelial and mesenchymal cells drives human dentinogenesis

doi: 10.1038/s41467-026-69545-3

Figure Lengend Snippet: a Split violin plots showing the expression distribution of WNT and NOTCH pathway components in DE (red) and DPL (blue). Each dot represents a single cell. Adjacent pie charts quantify the percentage of cells within the DE population expressing specific WNT and NOTCH ligands. b Dot plots illustrating the dynamic expression of WNT and NOTCH pathway genes in DE (left panel, red color scale) and DPL (right panel, blue color scale) across developmental stages. Dot size is proportional to the percentage of cells expressing the gene, and color intensity reflects the average z-score normalized expression level. PCW, post-conception weeks; Y, years. c Line graphs tracking the average expression of key WNT ( WNT6 , WNT7B , WNT10B ) and NOTCH ( JAG1 ) ligands within the DE population over time. d IF validation of the spatial localization of key WNT and NOTCH pathway proteins across different developmental stages. Target proteins (WNT6, WNT7B, WNT10B, DKK1, SFRP1, LRP6, LEF1, JAG1, NOTCH2, HEY1) are shown in green or red, with nuclei counterstained with DAPI (blue). Dotted lines outline the epithelial-mesenchymal boundary. Insets show magnified views of the indicated regions. Images are representative of experiments that were independently repeated three times with similar results ( n = 3 biological replicates). PT permanent tooth. Scale bars: 100 μm. Data in ( a – c ) are derived from scRNA-seq analysis of n = 2 biological replicates per developmental stage.

Article Snippet: Primary antibodies were applied against: DSPP (Abcam, ab216892), Ki67 (Cell Signaling Technology, 9129, Clone D3B5), KRT14 (Abcam, ab119695, Clone LL002), WNT6 (Abcam, ab50030), WNT7B (Proteintech, 10605-1-AP), WNT10B (Abcam, ab70816), LRP6 (Santa Cruz, sc-25317, Clone C-10), JAG1 (Abcam, ab300561, Clone EPR26388-51), NOTCH2 (Abcam, ab313453, Clone EPR28701-38), DKK1 (Abcam, ab61034, Clone EPR4759), SFRP1 (Proteintech, 26460-1-AP), HEY1 (Proteintech, 19929-1-AP), LEF1 (Abcam, ab137872, Clone EPR2029Y), BMP2 (Abcam,ab214821), BMP4 (Abcam, ab39973), GFP(CST, 2956, Clone D5.1) and rabbit IgG isotype control (CST, 3900, Clone DA1E) were added.

Techniques: Expressing, Single Cell, Biomarker Discovery, Derivative Assay