RAD21 Cooperates with Pluripotency Transcription Factors in the Maintenance of Embryonic Stem Cell Identity (original) (raw)
Figure 3
Depletion of RAD21 and other cohesin subunits leads to differentiation of ESCs.
(A): qPCR analysis of esiRNA knock-down efficiency for indicated genes (48 h RNAi, n = 3, error bars denote s.d. and *, ** indicate p<0.05 and 0.01 respectively). Transfection of a non-targeting esiRNA (Luc) was used as a control. Numbers adjacent to the gene names indicate independent, non-overlapping esiRNAs transfected. (B): Western Blot analysis of esiRNA knock-down efficiency for indicated genes (48 h RNAi; esi-1 and esi-2 indicate independent, non-overlapping esiRNAs transfected). Transfection of a non-targeting esiRNA (Luc) was used as a control. GAPDH served as a protein loading control. (C): Alkaline phosphatase staining of ESCs, which had been transfected with esiRNAs targeting RAD21, SMC1a and SMC3 (72 h post RNAi) showed a strong differentiation phenotype compared to a control (Luc). Nanog depletion served as a positive control for ESC differentiation. Scale bars correspond to 100 µm. (D): Quantification of alkaline phosphatase staining (n = 3) separated into undifferentiated (green), mixed (yellow) and differentiated colonies (red). (E+F): qPCR validation of expression changes of (E) stem cell maintenance genes and (F) lineage marker genes upon knock-down of RAD21 (two independent esiRNAs) and Luc control (48 h RNAi, n = 3, error bars denote s.d. and *, **, *** indicate p<0.05, 0.01 and 0.001 respectively).