Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2003 Aug 4;162(3):469–479. doi: 10.1083/jcb.200212067

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

Copyright © 2003, The Rockefeller University Press

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).

PMC Copyright notice

Figure 3. — Ceramide content and apoptosis during in vitro neuronal differentiation of ES cells. (A) Neutral lipids were purified from differentiating ES cells and a lipid amount corresponding to 750 μg of cellular protein/lane separated by HPTLC in the running solvent CH₃Cl/HOAc (9:1, vol/vol). Lipids were stained with the cupric acetate reagent. Ceramide (open bars) was quantified by densitometric analysis and comparison with known amounts of standard lipid (N-oleoyl sphingosine). Lane 1, ceramide standard from bovine brain; lane 2, fibroblast-freed ES cells, after 4 d in culture; lane 3, EBs at the EB4 stage; lane 4, EBs at the EB8 stage; lane 5, NPs at the NP2 stage; lanes 6–8, three terminal differentiation stages, 24 (D1 stage), 48 (D2 stage), and 96 h (D4 stage) on cultivation of NP cells in serum-containing medium; lanes 9–11, N-oleoyl sphingosine, 250, 500, and 1,000 ng, respectively. (B) Lipids were extracted from differentiated ES cells and the amount of ceramide quantified using the DAG kinase assay. Apoptosis (solid bars) was determined by TUNEL staining. For HPTLC, DAG kinase, and TUNEL analyses, experiments were performed with five independent ES cultures. The bars show the standard mean and deviation of percentage of TUNEL-positive cells that were counted in five areas of 200 cells in each experiment.

Figure 3. — Ceramide content and apoptosis during in vitro neuronal differentiation of ES cells. (A) Neutral lipids were purified from differentiating ES cells and a lipid amount corresponding to 750 μg of cellular protein/lane separated by HPTLC in the running solvent CH₃Cl/HOAc (9:1, vol/vol). Lipids were stained with the cupric acetate reagent. Ceramide (open bars) was quantified by densitometric analysis and comparison with known amounts of standard lipid (N-oleoyl sphingosine). Lane 1, ceramide standard from bovine brain; lane 2, fibroblast-freed ES cells, after 4 d in culture; lane 3, EBs at the EB4 stage; lane 4, EBs at the EB8 stage; lane 5, NPs at the NP2 stage; lanes 6–8, three terminal differentiation stages, 24 (D1 stage), 48 (D2 stage), and 96 h (D4 stage) on cultivation of NP cells in serum-containing medium; lanes 9–11, N-oleoyl sphingosine, 250, 500, and 1,000 ng, respectively. (B) Lipids were extracted from differentiated ES cells and the amount of ceramide quantified using the DAG kinase assay. Apoptosis (solid bars) was determined by TUNEL staining. For HPTLC, DAG kinase, and TUNEL analyses, experiments were performed with five independent ES cultures. The bars show the standard mean and deviation of percentage of TUNEL-positive cells that were counted in five areas of 200 cells in each experiment.