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. 2015 Mar 14;34(12):1630–1647. doi: 10.15252/embj.201489947

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© 2015 The Authors. Published under the terms of the CC BY NC ND 4.0 license

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

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A qRT–PCR assay of NMD target gene expression after reconstitution of Smg6^Δ/Δ ESCs with NMD-proficient or NMD-deficient Smg6 constructs (see Supplementary Fig S5A). The data are from three independent qRT–PCR experiments.

B Telomere FISH analysis of metaphases of ESCs with indicated genotype and reconstituted with indicated Smg6-expression vectors. The number of telomere-signal-free ends per metaphase is shown. n, the total number of metaphases used for the quantification. NMD+, NMD-proficient vectors; NMD-, NMD-deficient vectors; telos+, telomere-proficient vectors; telos-, telomere-deficient vectors. Statistic analysis was used to compare the Δ/Δ samples and their derived clones with different Smg6 truncation expression vectors.

C Spontaneous differentiation of Smg6^Δ/Δ (Δ/Δ) ESCs reconstituted with NMD-proficient (FL and ΔN) or NMD-deficient (Δ14-3-3 and ΔPIN) Smg6 constructs. Representative images of AP staining of cultures at day 6 after the removal of LIF and feeder are shown.

D EB formation assay of Smg6^Δ/Δ ESCs reconstituted with NMD-proficient or NMD-deficient Smg6 vectors. The morphology of EB cells and cavity formation at day 8 are indicative of the differentiation status.

E Immunofluorescence analysis of expression of the stem cell marker Oct4 on EBs of reconstituted Smg6^Δ/Δ ESCs on day 8.

F Western blotting of Smg6^Δ/Δ EB cells reconstituted with various Smg6 truncation vectors on day 8. The expressions of Oct4, Nanog, and c-Myc are shown. β-Tubulin was used as a loading control.

G Immunofluorescence analysis of expression of markers β-catenin and α-SMA on EBs of reconstituted Smg6^Δ/Δ ESCs on day 8.

H Chimerism analysis of in vivo rescue of differentiation defects of Smg6^Δ/Δ ESCs after reconstitution with NMD-proficient (FL-Smg6) or NMD-deficient (ΔPIN-Smg6) construct. Parental ES clone E22 (Smg6-CER) contained the Cre sequence, which was used to detect ESC contribution in E12.5 embryos by PCR. All ESC clones were pretreated with 4-OHT for 6 days to delete endogenous Smg6 before blastocyst injection.

Data information: The error bars represent the SEM. Unpaired Student's t-test was used. n.s., not significant; **P < 0.01. Source data are available online for this figure.