DEV02858 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. (A) Dose response of inhibition of Erk phosphorylation in increasing concentrations of PD184352. Cells were plated in differentiation conditions with the indicated concentrations of PD184352 or with DMSO alone as a vehicle and lysed daily for western blotting analysis. (B) Overexpression of MKP3 inhibits neural specification. 46C/T cells (Ying et al., 2003) were transfected with the plasmid pCAGMKP3mycIP encoding the Erk1/2 phosphatase MKP3 linked to the gene conferring puromycin resistance. Following selection in puromycin, cells were differentiated for 3 days and photographed for Sox1-EGFP. Scale bar: 200 μm. (C) PDK1-null cells can differentiate to nestin-expressing neural precursors. Monolayer differentiation was performed on wild-type and PDK1^−/− ES cells for 5 days before fixing and staining for nestin (DSHB, Iowa). Knockout cells can differentiate into nestin-positive neural precursors. Scale bar: 50 μm. (D) ES cells were differentiated for 2 days under conditions shown then fixed and immunostained for Fgf5 (sc-7914; Santa Cruz). Addition of 3 μM PD184352 does not inhibit FGF5 upregulation. Scale bar: 75 μm.

• Supplemental Figure S2 -

  Fig. S2. Cell cycle distribution of cells at day 2 of differentiation as assayed by flow cytometry. (A) Cells in N2B27; (B) cells in N2B27 plus 3 μM PD184352. Treatment with PD184352 does not cause cell cycle arrest. Shading indicates the populations of cells in G1 (green), S (olive) and G2/M (blue); the sum of these populations is shown in pink. The proportions of cells in each cell cycle stage were calculated using FlowJo (Treestar) and the Dean/Jett/Fox model.

  References

  Ying, Q. L., Nichols, J., Chambers, I. and Smith, A. (2003). BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell 115, 281-292.