Figure 1.
A temporal gene expression profiling of hESC differentiation.
A, Experimental representation of the time kinetic multilineage differentiation. B, An unsupervised hierarchical clustering analysis demonstrates that a temporal gene expression pattern changes during differentiation and the major changes were observed between days 12 to 14. Three independent biological experiments were performed for statistical analysis (p≤0.05). C, The dendrogams pattern of array cluster showed that a two major negative correlation for day 0 to 9 and 12 to 21 differentiation. D, The selected germ layer lineage specific BP (Enrichment p≤0.01) for up-regulated transcripts demonstrated that the numbers of genes are optimal between days 12 and 15. The figure represents (i) endoderm-specific, ii) ectoderm-specific and iii) mesoderm-specific BP. The x-axis represents the days of differentiation, and the y-axis represents number of transcripts.
Figure 2.
Enrichment of embryonic development associated genes in 14 days of hESC differentiation.
A, A schematic representation of the 14 days hESC differentiation. B, An unsupervised hierarchical clustering analysis of DEG in hESC differentiation showed two distinct clusters for up and down-regulated genes. C, The over expressed neuron development BP (from Table S4) encompasses 86 genes and their gene expression pattern showed in volcano plot. In x-axis represents fold change and y-axis showed FDR-controlled p-values (p≤0.05). D, The pluripotency markers were significantly downregulated (as shown by microarray) after 14 days of differentiation. E, The mass spectrometry analysis of regulated proteins unravelled the up-regulated spots for neuronal development related proteins and (F) the down-regulated spots for pluripotency regulators. The differentially regulated proteins (n = 3), (p≤0.01) are shown with their corresponding spot ID and UniProt ID (E (I), F (I)). Their expression patterns are represented as a percentage of volume (E (II), F (II)). The error bar represents SEM from 3 independent biological replicates.
Table 1.
The common differentially regulated gene (mRNA (n = 3), (p≤0.05) and protein (n = 3), (p≤0.01)) expression pattern after 14 days differentiation.
Figure 3.
Dose-dependent gene expression response of thalidomide in hESC differentiation.
A, The experimental approach of hESC differentiation and thalidomide treatment. B, Selected thalidomide concentrations do not induce any cytotoxicity in hESC differentiation. The scale bar represents 250 µm. C, 3D PCA showed that thalidomide treatment induces the changes in the gene expression pattern with increasing concentration. The 3 biological replicates for each sample group are showed in single colour. D, The number of DEG progressively increased with thalidomide concentration. Black shows up-regulated and grey shows down-regulated transcripts. Fold change ≥2 or ≤−2, (p≤0.05). E, An unsupervised hierarchical clustering of DEG demonstrated a dose-dependent repression. The highly expressed genes in untreated control were repressed for thalidomide in a concentration dependent manner. Data represents from 3 biological replicates. F, To quantify the BP the average relative fold change values were calculated manually (for all transcripts present in each GO) and represented for 14 day old EBs and thalidomide treatment. The error bar represents SEM from fold change values for transcripts belong to each GO.
Table 2.
Selected down-regulated significant (p≤0.01) GO categories after thalidomide treatment.
Figure 4.
Thalidomide perturbed heart, limb development and WNT signalling associated genes.
A, The heat map shows the microarray expression pattern for the selected genes for heart, limb development (from Table S6D, S6F) and WNT signalling (from Table S9). Since multiple genes are involved in various developmental processes few redundant genes are present. The up-regulated genes in control were repressed in a dose dependent manner. B, The representative heart (I), limb development (II) associated genes were analysed using RT-qPCR analysis with an independent experiment. Thalidomide responsive genes were shown in I and II (*p-value≤0.01, thalidomide-treated vs untreated 14-days old EBs) and 14 days old EBs gene expression were shown in (III) *p-value≤0.01, 14 days old EBs vs ESCs . The error bars represents the SEM from 3 technical replicates. C, The mass spectrometry results showed down-regulation of DDAH2 gene (n = 3), (p≤0.01). Figure C (I) represents spot analysis of DDAH2, mRNA and protein fold change (FC) values. C (II) The immunoblotting analysis of representative genes for thalidomide treatment. D, The immunoflourescence results shows the localisation of β-catenin and the gradient suppression at 70 µM thalidomide treatment. The scale bar represents 20 µm. E, The figure representing limb bud patterning was adapted from [40] and modified. The figure shows the molecular markers that SHH and ZPA (essential for limb development) were affected by thalidomide treatments. The green colour coded genes are more than −1.8 fold down-regulated.
Figure 5.
Thalidomide suppressed the GST genes and nucleocytoplasmic transporters at mRNA and protein level.
To validate microarray expression. A, (I) (RT-qPCR analysis) 14 day's hESC differentiation showed higher GST expression (*p-value≤0.01, 14 days old EBs vs ESCs) and (II) thalidomide treatment (**p-value≤0.05, thalidomide-treated vs untreated 14-days old EBs) repressed GST genes more than −2 fold change. The error bars represent the SEM from 3 technical replicates from an independent experiment. B, Immunoblotting analysis of GSTA1/2 confirms thalidomide induced perturbation at protein concentration. C, The cellular localisation of GSTA1/2 showed pronounced concentration of GSTA on nuclear membrane in control is attenuated for 10 and 70 µM. The scale bar represents 20 µm. D, The genomic and mass spectrometry analysis showed down-regulation of RANBP1. E, Investigation of transporters genes for thalidomide treatment with RT-qPCR analysis (from independent experiment). The error bars represent the SEM from 3 technical replicates. F, Thalidomide mediated protein concentration changes were shown with immunoblotting analysis. G, The sub cellular localisation of RANBP1 is demonstrated with immunoflourescence analysis. The cytoplasmic enrichment of RANBP1 gradient is repressed with thalidomide treatment. The scale bar represents 20 µm.