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. Author manuscript; available in PMC: 2010 Mar 1.
Published in final edited form as: Nature. 2008 Mar 23;453(7192):223–227. doi: 10.1038/nature06863

Figure 1. REST regulates self-renewal in mouse ES cells.

Figure 1

a, EB cells (ES cells grown in the absence of LIF under nonadherent conditions for 4 d) showed reduced levels of self-renewal markers and REST compared with ES cells (grown in the presence of LIF under adherent conditions). Western blot analysis of whole-cell extracts prepared from ES cells and EB cells showed an association between expression of REST and self-renewal markers. Actin was used as a loading control. b–f, Mouse ESC lines with heterozygous deletion of REST (RRC and YHC) show loss of self-renewal. b, RT-PCR analysis and c, Q.RT-PCR analysis with different primer sets of total RNA isolated from ES, RRC, and YHC cells showed reduced REST transcripts in REST+/− cells. Gapdh was used as a loading control. * p < 0.0001. The values are represented as mean +/− SD (n=3). d, Western blot analysis of whole-cell extracts from ES, RRC, and YHC cells showed reduced REST protein in REST+/− cells. α-Tubulin was used as a loading control. e, Alkaline phosphatase staining of ES colonies showed loss of self-renewal in REST+/− cells as compared with wild-type ES cells f, Percentages of self-renewing colonies of ES, RRC, and YHC cells calculated after alkaline phosphatase assays when cultured under self-renewing conditions showed significant reductions in the self-renewal capacity of both REST+/− cell lines compared with ES. * p < 0.0001. The error bars correspond to three replicates (n=3). g–i, siRNA-mediated knockdown of REST caused loss of self-renewal in mES cells. g, Specific knockdown of targeted genes was achieved using siRNA. Q.RT-PCR of total RNA purified from mES cells treated with siREST or siOct4 showed knockdown of specific genes. Analysis was performed 5 d after transfection. * p < 0.0001. The values are represented as mean +/− SD (n=3). h, Western blotting showed reduced REST protein levels in siREST treated cells compared with control (NT siRNA). i, siREST- and siOct4-treated cells showed less self-renewal than NT-treated cells did. mES cell colonies were screened by alkaline phosphatase assays. * p < 0.0001; ** p < 0.001. The error bars correspond to three replicates (n=3). j, Exogenously added REST, but not GFP, maintained self-renewal in mES cells cultured under differentiation conditions. mES cells were transfected with plasmids encoding GFP or REST and grown in the absence of LIF. Percentages of self-renewing colonies from three independent experiments were averaged after alkaline phosphatase assay and are shown for each transfected gene. *** p < 0.01. The error bars correspond to three replicates (n=3).