Figure 1. Smg6-deficient ESCs fail to differentiate in vitro and in vivo.

A In vitro culture of control (+/Δ) and Smg6^Δ/Δ (Δ/Δ) blastocysts. Note that there is no ICMs outgrowth of Smg6^Δ/Δ blastocysts on day 5 of culture in contrast to the controls.

B Morphology of control (F/F) and Smg6^Δ/Δ ESCs.

C Proliferation curve of control (F/F) and Smg6^Δ/Δ ESCs.

D Cell death analysis of control (F/F) and Smg6^Δ/Δ ESCs by FACS after Annexin-V staining (n = 3 for each genotype).

E H&E staining of control and Smg6^Δ/Δ ESCs derived EBs on day 8. Enlarged panels (right) show that control EBs contained differentiated connective tissues and epithelial-like structures and Smg6^Δ/Δ EBs contained undifferentiated cells.

F Immunostaining and quantification (top and lower left) and Western blotting (lower right) of Oct4 expression in control and Smg6^Δ/Δ ESCs derived EBs on day 8. β-Tubulin was used as a loading control. n, the total number of cells counted from five controls and six Smg6^Δ/Δ EBs.

G Immunostaining of ectoderm marker Nestin, endoderm marker β-catenin, and mesoderm marker α-SMA on day 8 of control F/F and Δ/Δ EB samples.

H ESC differentiation upon DMSO and RA treatment. Note that Smg6^Δ/Δ ESCs continued to maintain ES-like structure, while control ESCs differentiated into different cell types (fibroblast-like cells by DMSO induction; endothelial cells after RA treatment) on day 5 after plating onto gelatin-coated dishes. The morphology of the EBs before plating was shown (day 0).

I Immunostaining of Oct4 expression in control and Smg6^Δ/Δ ESCs derived differentiation cultures treated for DMSO or RA on day 8. The frequency of Oct4⁺ cells was summarized in low panels. n, the total number of cells used for the quantification. At least three differentiation cultures were used.

J Chimerism assay of the contribution of GFP-labeled control and Smg6^Δ/Δ ESC derivatives to different mouse tissues (E12.5 embryos are analyzed). Note the absence of GFP-labeled Smg6^Δ/Δ ESC derivatives within chimeras. Representative images of the brain (ectoderm), liver (endoderm), and skin connective tissues (mesoderm) are shown.

Data information: The error bars represent the SEM. Unpaired Student's t-test was used. ***P < 0.001.Source data are available online for this figure.