Colony Formation Assay Colony formation assays were performed to investigate the effects of Gremlin overexpression on cell proliferation. Cells (800 cells/well) were seeded onto 6-well plates for 10 days. Culture medium was changed twice a week. After 10 days incubation, the colonies were washed with 1xPBS and fixed with methanol. For the colony formation assay, cells were fixed with methanol and stained with crystal violet (in 25% methanol) for 30 minutes at room temperature. Supporting Information Figure S1 Effect of Grem1 overexpression on NL-20 and HFL-1 cell proliferation. (A) Gremlin protein expression by immunoblot analysis. (B) Colony formation assays documenting the effect of Grem1 overexpression on the growth of NL-20 and HFL-1 cells. Cells (1,000/well) were seeded onto 6-well plates and maintained for 10 days. (TIF) References 1. Weinberg RA (1994) Oncogenes and tumor suppressor genes. CA: A Cancer Journal for Clinicians 44: 160–170. 2. 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