Figure 1.
HPRT knockdown in murine ESD3 cells.
(A) Reduction of HPRT mRNA expression as assayed by qPCR; (B) reduction of immunoreactive HPRT protein as determined by Western blot analysis; (C) reduction of HPRT enzymatic activity as measured by absent detectable IMP production as detected by autoradiography of a thin layer chromatography of cell lysate (6).
Figure 2.
Neuron-like cells generated from WT control and HPRT-knockdown ESD3 cells.
Figure 3.
Regulation of pluripotency markers at the embryonic stem cell, SNM and neuronal (DA) stages of neuronal differentiation of WT control and HPRT-knockdown ESD3 cells, as measured by qPCR analysis.
(A) Expression of Oct-4, showing expected down regulation; (B) expression of Nanog during differentiation; (C) expression of Sox2 during differentiation. Control cells show the expected down regulation at the SNM and DA stages but HPRT-knockdown cells show up-regulated expression during both SNM and DA stages of differentiation; (D) expression of the neuronal marker β-III tubulin showing the expected up-regulation in control cells, but reduced expression during differentiation of HPRT knockdown cells; (E) up-regulation of the dopaminergic neuronal marker DAT in control cells and HPRT-knockdown cells.
Figure 4.
Immunohistochemical detection of the neuronal marker β-III tubulin and tyrosine hydroxylase (TH) in ESD3 cells at the final 14-day neuronal stage of SNM differentiation.
(A) β-III tubulin in WT control cells; (B) TH expression in control WT cells; (C) merged β-III tubulin and TH expression in WT control cells; (D) β-III tubulin in HPRT-knockdown cells; (E) TH in HPRT-knockdown cells, and (F) merged β-III tubulin and TH expression in HPRT-knockdown cells.
Figure 5.
FACS analysis of control WT (A) and HPRT-knockdown (B) cells expressing TH.
The cell types demonstrate indistinguishable high percentages of TH-positive cells.
Table 1.
Number of genes found by microarray analysis to be differentially expressed in HPRT-knockdown ESD3 cells at the ES, SNM, and DA neuronal stages of differentiation.
Table 2.
GO functions significantly altered (p≤0.05) in the GO term “nervous system” at the SNM stage of control and HPRT-knockdown cells.
Table 3.
GO functions significantly altered (p≤0.05) in the GO term “nervous system” at the DA neuronal stage of control and HPRT-knockdown conditions.
Figure 6.
Western blot analysis for glial markers MBP and Olig2 of control WT cells and HPRT-knockdown ESD3 cells at the final neuronal stage of differentiation.
Both markers demonstrate a significant increase of expression in the HPRT-knockdown cells.
Table 4.
Significantly altered (p≤0.05) myelination-related pathways in HPRT knockdown cells.
Table 5.
Expression of “dopamine” GO terms in differentiated neuronal cells.
Table 6.
Significantly altered (p≤0.05) GPCR signaling pathways in control and HPRT-knockdown cells.
Table 7.
Significantly altered (p≤0.05) signaling pathways related to GO term “neurodegenerative disease” in control and HPRT-knockdown cells.
Figure 7.
Mean shift and polarization of canonical pathways.
Gene expression of 802 canonical pathways was tested for (A) shift in mean expression and (B) polarization, each test aggregating paired differences between wild-type (WT) and HPRT-knockdown (KD) at each time point during the differentiation. Rows are significant pathways, organized by source database and with the top three WT and KD genes provided underneath the pathway name. Columns are the module response over time, with red/blue indicating over-expression in WT/KD cells and diameter indicating the fraction of genes in the pathway with differential expression. The mean-shift test statistic is the mean log-ratio of WT to KD expression aggregated over time points for genes within a pathway; the polarization test statistic is the variance of gene log-ratios within a pathway. For both tests, the null distribution is obtained by permutation of sample labels. Thresholds are two false discoveries (FDR = 0.22) for mean shift and one false discovery (FDR = 0.042) for polarization.