Figure 1.
Comparative analysis of apoptosis-related genes expression profiles in undifferentiated ESCs and maGSCs.
(A) The format of the Mouse Apoptosis OligoGEArray spotted with oligos against 112 genes known to be involved in apoptosis-related processes. Additionally, oligos spotted against housekeeping genes (red) and blank or plasmid controls (blue) are also shown. (B) OligoGEArray blot showing the expression profile of apoptosis-related genes in undifferentiated ESCs and maGSCs of the 129Sv genetic background. (C) Scatterplot analysis of genes expressed in undifferentiated ESCs and maGSCs of the 129Sv background. Expression of pro-apoptotic gene Bok and three anti-apoptotic genes (p53, Birc2, and Birc5) were upregulated in maGSCs relative to ESCs, but the expression of most genes did not substantially differ between the cell types. (D) OligoGEArray blot showing the expression profile of apoptosis-related genes in undifferentiated ESCs and maGSCs of the Stra8-EGFP transgenic background. (E) Scatterplot analysis of genes expressed in undifferentiated ESCs and maGSCs of the Stra8-EGFP background. Two pro-apoptotic genes, Bnip3 and Tnfsrf12a, were slightly downregulated in maGSCs relative to ESCs, but most genes did not substantially differ between the cell types.
Figure 2.
Expression profiling of apoptosis-related genes in differentiated ESCs and maGSCs.
(A) OligoGEArray blot showing the expression pattern of apoptosis-related genes in ESCs and maGSCs of the 129Sv background that had been differentiated for 21 days with retinoic acid. (B) Scatterplot analysis of differentiated ESCs and maGSCs revealing similar gene expression patterns in both differentiated cell types with upregulation of Nfkb1 and downregulation of Pycard and Bnip3 in differentiated maGSCs. (C) Heatmap analysis of undifferentiated ESCs and maGSCs (from both the 129Sv and the Stra8-EGFP backgrounds) as well as differentiated ESCs and maGSCs, revealing all undifferentiated cell types in one cluster, while differentiated cell types are distant and clustered together. (C′) Specific and strong expression of several anti-apoptotic and pro-apoptotic genes in undifferentiated ESCs and maGSCs relative to differentiated cells was highlighted.
Figure 3.
Induction of apoptosis and transcriptome analysis of early-apoptotic ESCs and maGSCs.
ESCs (A) and maGSCs (B) treated with DMSO for 24 h showed typical colony morphology, whereas cells treated with CTN for 24 h lost their typical colony morphology and appeared as blebs. (A′) Stacked bar graph showing the flow cytometric data of annexin-V and 7-AAD staining on control (non-treated), DMSO-treated, and CTN-treated ESCs. (B′) Stacked bar graph showing the flow cytometric data of annexin-V and 7-AAD staining on control (non-treated), DMSO-treated, and CTN-treated maGSCs. (C) Outline of the strategy to identify pluripotency- as well as apoptosis-related genes in pluripotent cells. ESCs and maGSCs were treated with CTN, and early apoptotic cells (annexin-V+ve/7-AAD−ve cells) were collected by flow cytometry. The sorted early-apoptotic cells were then used for transcriptome analysis. (D) The transcriptomes of early-apoptotic ESCs and maGSCs were ∼94% identical during the apoptotic response, whereas 6% genes were differentially expressed. (E) Principle component analysis showing the clustering of early apoptotic ESCs and maGSCs transcriptomes, which are distant to previously generated transcriptomes of undifferentiated as well as differentiated ESCs and maGSCs.
Table 1.
List of GO terms associated with upregulated genes in early-apoptotic cells.
Table 2.
List of GO terms associated with downregulated genes in early-apoptotic cells.
Figure 4.
Identification and characterization of novel pluripotency- and apoptosis-related genes.
(A) Heatmap representation of qPCR data of selected candidate genes in ESCs (ES RI) and adult mouse tissues. (B) The qPCR analysis confirming the downregulation of Fgf4 in early apoptotic ESCs and maGSCs. (C) The qPCR analysis confirming the downregulation of Mnda in early apoptotic ESCs and maGSCs. (D–H) Stacked bar graphs showing the percentage of viable, early-apoptotic, and late-apoptotic cells in either DMSO- or CTN-treated cells. Induction of apoptosis and analysis by annexin-V/7-AAD staining after 12 h and 24 h of CTN treatment in wild-type ESCs (D), Fgf4-OE (Fgf4-overexpressing) cells (E), Fgf4-KO (Fgf4-knock-out) cells (F), Mnda-OE (Mnda-overexpressing) cells (G), Mnda-DN (Mnda-downregulated) cells (H). The flow cytometry data of three or more independent biological replicates were calculated and represented as a mean ±SD. The data was analyzed for statistical significance and found no significant differences in D–H.
Figure 5.
Role of Fgf4 and Mnda during genotoxic stress response of ESCs.
(A) Stacked bar graphs showing the percentage of cells at various stages of cell cycle (SubG1/Apoptotic, G1, S and G2/M) in untreated, Control ESCs – wildtype ESCs; Fgf4-OE – Fgf4 overexpression cells; Fgf4-KO – Fgf4 knockout cells; Mnda-OE – Mnda overexpression cells; Mnda-DN – Mnda downregulation cells. (B–F) Genotoxic stress was induced by treatment with NCS for 30 min followed by recovery for indicated time points and analyzed for cell cycle parameters in Control ESCs – wildtype ESCs; Fgf4-OE – Fgf4 overexpression cells; Fgf4-KO – Fgf4 knockout cells; Mnda-OE – Mnda overexpression cells; Mnda-DN – Mnda downregulation cells. The cell cycle data of three or more independent biological replicates were calculated and represented as a mean ±SD. The values which are statistically significant are indicated with asterisks (∗p<0.05).