Fig. 5.
Overexpressed POLR3G or POLR3GL in Polr3gΔ/Δ ESCs equally rescue the phenotype of Polr3gΔ/Δ ESCs. (A) Immunoblots (Top five panels) of whole-cell extracts of ESCs with different genotypes (indicated at the top) with POLR3C, POLR3D, POLR3F, POLR3G, and POLR3GL antibodies (indicated at the left). Each lane was loaded with the same amount of protein extract as indicated by Coomassie blue staining at the bottom. (B) Cell proliferation assays of wild-type, Polr3gΔ/+, and Polr3gΔ/Δ ESCs. Two Polr3gΔ/Δ stable ESC lines were used in this assay as shown by the green and purple lines. (C–G) Differentiation assays performed with wild-type, Polr3gΔ/+, and Polr3gΔ/Δ ESC lines and with rescue lines Polr3gΔ/Δ;EF1a-3g and Polr3gΔ/Δ;EF1a-3gl. RT-PCR assays were performed on Oct4, Nanog, Gata4, Gata6, and Nestin genes using three time points (ESCs at the differentiation start point, 3 d after EB formation and 6 d after EB formation) individually. (H) Embryoid body differentiation analyses indicating that overexpression of either POLR3G or POLR3GL rescues the undifferentiated phenotype of Polr3gΔ/Δ ESCs.