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Blood, Vol. 114, Issue 1, 60-63, July 2, 2009
Expression of Scl in mesoderm rescues hematopoiesis in the absence of Oct-4
Blood Kong et al.
114: 60
Supplemental materials for: Kong et al
Files in this Data Supplement:
- Table S1. Primers used for the RT-PCR analyses (PDF, 49 KB)
- Figure S1. Formation of hematopoietic progenitors from day 14 EBs derived from wildtype CGR8 and ZHBTc4 ESCs (JPG, 629 KB)
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(A) Parental CGR8 and conditional Oct-4 knockout ZHBTc4 ESCs were cultured in methylcellulose medium supplemented with hematopoietic cytokines in the absence of LIF. (B) RT-PCR expression analyses of a time course of mesodermal and hematopoietic gene expression in differentiating ZHBTc4 and CGR8 ESCs/EBs with and without tet. Representative PCR data is shown. Each PCR was repeated at least 3 times with cDNA prepared from independent ESC differentiations. ESCs = embryonic stem cells. EBs = embryoid bodies. Untx = untreated.
- Figure S2. Biochemical analyses on SCL stably-transfected ESC clones when cultured in undifferentiated or differentiated conditions (JPG, 777 KB)
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(A) SCL expression from control undifferentiated ESC clones (CAPP) and undifferentiated SCL transfected clones (SCL/CAPP) was assayed by RT-PCR. Amplification of 18S rRNA is shown as a loading control. (B) Immunoblot analysis of SCL and Oct-4 expression from undifferentiated control and SCL ESCs. β-actin protein expression is shown to demonstrate equal loading of lanes. (C) RT-PCR analyses of SCL expression from undifferentiated tet-treated control and tet-treated SCL ESC clones. (D) Immunoblot analysis of SCL and Oct-4 protein from undifferentiated tet-treated control and tet-treated SCL ESC clones. (E) Immunoblot analysis was performed with whole cell extracts prepared from WT CGR8, WT RW4, ZHBTc4 control (CAPP-3), and SCL ESCs (SCL-10) induced to differentiate into EBs for 0 to 6 days. Immunoblots were incubated with antibodies to SCL, Oct-4, and β-actin. SCL antibody at 1:500, Oct-4 antibody at 1: 400 and actin antibody at 1;10,000. CAPP: parental vector stably-transfected wild type ESC clones. SCL/CAPP: SCL stably-transfected ESC clones. Cln = clone. Untx = untreated. Tet = tetracycline. D = day. Both CAPP control and SCL clones are ZHBTc4-derived Oct-4 knockout ESCs with tet-regulated Oct-4 rescue.
- Figure S3. RT-PCR analyses of mesodermal and hematopoietic gene expression in day 0 to day 6 control and SCL EBs (JPG, 543 KB)
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Control (CAPP-3) or SCL-expressing (SCL-10) ESCs were treated with tet at the beginning of differentiation induction. RNA was collected at days 0, 2, 4, and 6 post-differentiation. Untx = untreated. D = day. Both CAPP control and SCL clones are ZHBTc4-derived Oct-4 knockout ESCs with tet-regulated Oct-4 rescue.
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