Fig 2: FOXM1 promotes RPE epithelial fate.A. qPCR based measurement of transcript expression of a panel of epithelial (red) and mesenchymal (green) markers at Day 10 post siFOXM1 transfection (except levels of FOXM1 itself which are measured at Day 2 post knockdown). Data is normalized to transfection with non-targeting siRNA used as a control. ACTB, GAPDH, IPO8 and HPRT1 are used as housekeeping genes. Bars represent Mean + SD (n = 3). P<0.05 (Student’s t-test). B. Immunocytochemistry for PMEL17 upon FOXM1 knockdown (siFOXM1) or overexpression (pFOXM1) at Day 10 post transfection. C. Level of knockdown obtained upon transient transfection of siRNA against SNAI2, SNAI1, ZEB1, TWIST1 and GSC. Knockdown was measured by qPCR at Day 6 post transfection and is expressed relative to non-targeting siRNA used as control. CYC1 and GAPDH were used as housekeeping genes. Bars represent Mean ± SD (n = 6–9). Knockdown of EMT-TF expression was significant, P<0.05 (Student’s t-test). D. No significant effect on PMEL, MITF or BEST1 expression was observed under the same conditions described above for Fig 2C.

Fig 3: FOXM1 regulates RPE proliferation.A. Graph showing quantification of immunocytochemistry where % Ki67 (n = 3) or % EdU (n = 6) is plotted on the left Y axis and relative expression of FOXM1 transcript (n = 3; ACTB used as housekeeping gene) on the right Y axis over days in culture (x axis). B. Quantification of change in FOXM1 transcript upon transient overexpression (pFOXM1) or knockdown (siFOXM1), 48h post transfection, measured by qPCR. Data is normalized to appropriate controls (Empty vector for pFOXM1 and non-targeting siRNA for siFOXM1). Bars represent Mean + SD (n = 3). C. Quantification of change in EdU incorporation 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). D. Quantification of immunocytochemistry for Ki67 upon siRNA mediated knockdown of non-targeting control, GAPDH, SNAI1, SNAI2 and FOXM1, at Day 6 post transfection. Bars represent Mean + SD (n = 3). n.s non-significant, * p<0.05 Student’s t-test. E. Effect of Thiostrepton on EdU incorporation [left Y axis, red] and FOXM1 transcript expression measured by qPCR [right Y axis, blue]. Bars represent Mean ± SD (n = 6). F. Bright-field microscopy showing a scratch introduced in a RPE monolayer at 0 hrs and 19hrs in the presence of DMSO or 10μM Thiostrepton. Edge of the scratch is marked with a white line. Scale bar = 200 μm. G. Quantification of F (above). Bars represent Mean + SD (n = 7). P<0.0001 (Student’s t-test)

Fig 4: Model showing proposed roles of FOXM1 in epithelial fate acquisition.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.

Fig 5: Epithelial fate acquisition is density dependent.A. Quantification of change in cell density (number of DAPI positive nuclei per cm2 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/cm2) or low (8000 cells/cm2) 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.