Supplementary Figure S1: Inhibition of FGF Signaling Decreases Sox2 Expression in Osteosarcoma

Supplementary Figure S1: Inhibition of FGF signaling decreases Sox2 expression in osteosarcoma cells

Western blot of mOS-482 cells treated either with DMSO or PD173074 for 48 hours.

Supplementary Figure S2: Sox2 depletion leads to a slightly lower rate of proliferation in osteosarcoma cells without increased senescence or apoptosis

mOS-482 cells stably expressing either scrambled or Sox2-specific shRNAs were analyzed for proliferation (A), and BrdU incorporation (B). Senescence and apoptosis were assessed by Senescence-associated-β galactosidase (SABG) Staining (C), and expression of cleaved caspase 3 (D).

Supplementary Figure S3: Sox2 depletion does not alter the ability of osteosarcoma cells to adhere to substrata

Fifty thousand osteosarcoma cells were plated per well in 96-well plates and then washed with PBS and fixed at 30-min intervals. Adherent cells were stained with 0.2% crystal violet in 20% methanol and washed with PBS to remove residual crystal violet. Crystal violet incorporation by adherent cells (a measure of cell adherence to substratum) was measured by lysing the cells in 2% SDS solution and reading the absorbance of each well at 570 nm. Data presented is the mean from 9 wells + S.D.

Supplementary Figure S4: p53 and Rb protein expression in tumors derived from mOS-482 cells, expressing either scrambled, or Sox2-specific shRNA (sh900 and sh1548)

Lysates from tumors derived from cells expressing either scrambled or Sox2 specific-shRNA (sh900 and sh1548) were run on an 8% SDS-PAGE gel and analyzed for expression of p53 and Rb by Western blotting. Positive Control = Wild-type osteoblasts. Rare tumors from cells expressing Sox2 shRNAs appeared with a long latency of over 12 weeks, while tumors from scrambled cells grew in 2 weeks. Lack of p53 and Rb protein expression confirms that tumors were derived from the originally injected p53-/-Rb-/- cells.

Supplementary Figure S5: FSC-SSC (Forward Scatter- Side Scatter) of cells used in analysis in Figure 3A

FSC-SSC (Forward Scatter-Side Scatter) profiles of cells singly labeled with PE-Sca-I (1), singly labeled with AF488-Sox2 (2), and doubly labeled with PE-Sca-I and AF488-Sox2 (3). To exclude debris, only cells within circular areas in FSC-SSC plots were used in analysis.

Supplementary Figure S6: Purity of Sca-IHI and Sca-ILO separated fractions used in Figure 3B

Dot plots of isotype-PE control (A), PE-Sca-I stained total cell population before separation (B), Sca-IHI (C), and Sca-ILO (D) fractions post separation. (E) Growth curve of Sca-1HI Sox2HI and Sca-1LO Sox2LO cells. *=p<0.05

Supplementary Figure S7: Sox2 over-expression increases osteosphere formation and adipogenic capacity of primary osteoblasts.

(A) Western analysis and osteosphere formation assay of primary murine osteoblasts transduced with FUCRW (control) or Sox2 lentivirus. * = P<0.05

(B) Decreased osteogenesis and increased adipogenesis in primary osteoblasts over-expressing Sox2 (Magnification: 100X for Adipogenesis Assay)