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

a, EB cells (ES cells grown in the absence of LIF under non-adherent conditions for 4 days) 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 ES cell lines with heterozygous deletion of Rest (RRC and YHC) show loss of self-renewal. b, c, RT–PCR (b) and qRT–PCR (c) analyses with different primer sets of total RNA isolated from ES, RRC and YHC cells show reduced Rest transcripts in Rest^+/- cells. Gapdh was used as a loading control. *, P < 0.0001. The values are represented as mean  s.d. (n = 3). d, Western blot analysis of whole-cell extracts from ES, RRC and YHC cells shows reduced levels of REST protein in Rest^+/- cells. -Tubulin was used as a loading control. e, Alkaline phosphatase staining of ES colonies shows 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 show significant reductions in the self-renewal capacity of both Rest^+/- cell lines compared with ES cells. *, P < 0.0001. The error bars correspond to three replicates (n = 3) and show mean  s.d. g–i, siRNA-mediated knockdown of REST causes loss of self-renewal in mouse ES cells. g, Specific knockdown of targeted genes was achieved using siRNA. qRT–PCR of total RNA purified from mouse ES cells treated with siRest or siOct4 shows knockdown of specific genes. Analysis was performed 5 days after transfection. *, P < 0.0001. The values are represented as mean  s.d. (n = 3). h, Western blotting shows reduced REST protein levels in siRest-treated cells compared with control (NT siRNA). i, siRest- and siOct4-treated cells show less self-renewal than NT-siRNA-treated cells. Mouse ES cell colonies were screened by alkaline phosphatase assays. *, P < 0.0001; **, P < 0.001. The error bars correspond to three replicates (n = 3) and show mean  s.d. j, Exogenously added Rest, but not GFP, maintained self-renewal in mouse ES cells cultured under differentiation conditions. Mouse ES 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) and show mean  s.d.