Table 1.
Cells used in this study.
Figure 1.
Selection of optimal reference genes: PGK1 and PPIA.
15 genes were evaluated for their appropriateness for use as reference genes to normalize all subsequent data. Using a method described by Vandesompele et al. [8] genes were ranked by a stability coefficient M and those genes having the lowest stability (greatest value of M) were eliminated from the subsequent round of stability calculations. The gene eliminated from each round is indicated in parentheses.
Figure 2.
Cell lines express appropriate markers of pluripotency and lineage.
Abundance of gene transcripts were determined by qPCR, and is expressed in linear arbitrary units. A. Expression of NANOG, a marker of pluripotency. B. Expression of SOX2, a marker of pluripotency and neural precursor cells. C. Expression of the microtubule-associated protein MAP2, a marker of neural cells. D. Expression of the transcription factor GATA6, a marker of heart and endoderm-derived tissues.
Figure 3.
hESC lines BG01 and TE06 express protein markers of pluripotency.
Antibodies against protein markers of pluripotency were used to label hESCs via immunofluorescence. Fixed TE06 (A, C, E) or BG01 (B, D, F) cells were incubated with primary antibodies directed against CD9 (A, B), Oct4 (A, B, E, F), E-cadherin (C, D), Nanog (C, D), or PODXL (E, F) followed by incubation with Alexa fluor 488 or 555 conjugated secondary antibodies. Pictures are 100× wide-field images captured on an inverted fluorescence microscope.
Figure 4.
A cohort of pro-apoptotic BH3-only BCL-2 family members are expressed primarily in hESCs.
Abundance of gene transcripts were determined by qPCR, and is expressed in arbitrary units. A. NOXA, B. BIK, C. BIM (assay detected all three transcript variants: BIM-L, BIM-EL, and BIM-S), D. BMF, E. PUMA, F. BNIP3. Asterisks indicate values that differ significantly from the average hESC expression level (p<0.001, two-tailed student's t test).
Figure 5.
Expression of pro-survival BCL-2 family members.
Abundance of gene transcripts were determined by qPCR, and is expressed in linear arbitrary units. A. BCL-2, B. BCL-w, C. BCL-x (assay detected both transcript variants: BCL-xS and BCL-xL), D. A1, E. MCL-1. Asterisks indicate values that differ significantly from the average hESC expression level (p<0.001, two-tailed student's t test).
Table 2.
BCL-2 family members with hESC or non-hESC expression bias.
Figure 6.
Hierarchical cluster analysis of BCL-2 family members in hESCs and non-hESCs.
qPCR data was clustered via a two-way unsupervised clustering algorithm (Cluster 3.0) and visualized as a heat map (Java TreeView 1.1). Parallel cultures of each cell line (number): TE06-NSCs (4), TE06 MAT (4), TE06 MEF (4), BG01 MEF (4), BG01 MAT (4), HEMn-LP (3), HEK (3), HMVEC (2), HDF (3), HUVEC (3), HPASMC (3), HMEC (3), MCF-7 (3), and HeLa (3). For many genes, assays detected all or a subset of the transcript variants. 1Variants 1–7. 2Variants alpha, beta, delta, and sigma. 3Variants alpha and delta. 4Varient sigma. 5BIM-L, BIM-EL, and BIM-S. 6BIM-L, BIM-S. 7BIM-S. 8Variant 5 9Variant alpha. 10Variants alpha and beta. 11Variant 1. 12Variant 2. 13BCL-xL and BCL-xS. 14Variant 3. 15Variants 1 and 2.