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. 2024 Feb 26;13:e94694. doi: 10.7554/eLife.94694

Figure 5. Centrioles in Sass6 em5/em5 mouse embryonic stem cells (mESCs) are formed de novo during derivation from blastocysts and are lost upon differentiation.

(A) Whole-mount immunostaining for TUBG on Cetn2-eGFP and Sass6em5/em5 Cetn2-eGFP blastocysts at E3.5. Trophoblasts (top) and inner cell mass cells (bottom) are demarcated by the dashed line. The Inset is a magnification of the dashed square. Scale bars = 5 µm and 1 µm (inset). (B) Quantification of the percentage of cells with centrioles (TUBG and Centrin-eGFP) from E3.5 blastocysts in (A). Three blastocysts per genotype were used for the quantifications. Error bars represent mean ± SD WT: 73 ± 11% (n=200 cells); Sass6em5/em5: 0 ± 0% (n=175). ***p<0.001, (two-tailed Student’s t-test). (C) Whole-mount immunostaining as mentioned in (A) on blastocysts after 24 hr in culture. (D) Quantification from (C) as mentioned in (B). Four blastocysts per genotype were used for the quantifications. WT: 98 ± 30% (n=630 cells); Sass6em5/em5: 33 ± 11% (n=690). ****p<0.0001. (E) Immunostaining for TUBG and FOP in WT and Sass6−/− cells after in vitro neural differentiation (NPCs). Insets are magnifications of the center of the dashed squares. Scale bars = 5 µm and 1 µm (insets). (F) Quantification of the percentage of cells with centrosomes (TUBG and FOP) in (E) from five independent experiments. Error bars represent mean ± SD WT: 97 ± 0% (n=1388 cells); Sass6−/−: 6 ± 0% (n=1068). ****p<0.0001, (two-tailed Student’s t-test).

Figure 5.

Figure 5—figure supplement 1. Wild-type (WT) and Sass6−/− mouse embryonic stem cells (mESCs) differentiated into neural progenitor cells (NPCs).

Figure 5—figure supplement 1.

(A) Immunostaining for NESTIN on WT and Sass6−/− cells after in vitro neural differentiation of mESCs into NPCs. Scale bar = 20 µm. (B) Immunostaining for TUBG and FOP in WT and Sass6−/− cells after in vitro neural differentiation (NPCs). Examples of cells with or without (in Sass6−/−) centrosomes are shown. Insets are magnifications of the center of the dashed squares. Scale bars = 5 µm and 1 µm (insets).