Figure 6.

Loss of BAP1 induces a dedifferentiated, stem-like phenotype in uveal melanoma cells. (a) RNA expression of melanocyte differentiation markers in BAP1-deficient or control 92.1 stable cells, primary melanocytes, and primary class 1 tumor cells. Fold change is compared to control knockdown cells. (b) Cell morphology of BAP1-deficient or control primary uveal melanocytes. (Left panel) representative pictures. (Right panel) dendritic arborizations measured as the number of branch points per cell, with a bipolar cell having zero aborizations; shown as percent of total cells. (c) Cell spindle morphology of BAP1-deficient or control primary uveal melanocytes after four weeks of shRNA expression, measured as the ratio of cell length to cell width. (d) RNA expression levels of stem cell genes NANOG and OCT4 in BAP1-deficient or control 92.1 and OCM1A stable cells. Fold change is compared to control knockdown cells. (e) Clonogenic assay using BAP1-deficient or control OCM1A stable cells. One cell/well was grown in serum-free stem cell media in non-adherent 96-well plates. The number of wells producing colonies was measured after 5 days. Data is shown as fold change over control. (f) Assay measuring the ability of BAP1-deficient or control stable cells to grow in stem cell culture conditions on non-adherent plates in serum-free media. Colony size was measured after 5 (OCM1A) or 7 (92.1) days using ImageJ software. (g) RNA expression of melanocyte differentiation markers in BAP1-deficient 92.1 stable cells after 72 hr treatment with 0.5 mM, 1.0 mM, or 2.0 mM HDAC inhibitor (HDACi) as indicated. Fold change is compared to control knockdown cells.