Expression of CoAA in embryoid body of P19 cells and in brain of mouse embryo

<p><b>Copyright information:</b></p><p>Taken from "Switched alternative splicing of oncogene CoAA during embryonal carcinoma stem cell differentiation"</p><p></p><p>Nucleic Acids Research 2007;35(6):1919-1932.</p><p>Published online 1 Mar 2007</p><p>PMCID:PMC1874587.</p><p>© 2007 The Author(s)</p> () Evaluation of anti-RRM and anti-CoAA antibodies using western blot analysis by overexpression of CoAA and CoAM in 293 cells. () Light microscopy of P19 cells at indicated differentiation stages (×400). () Embryoid body at Day 4 was paraffin-embedded, sectioned and stained with anti-RRM (against both CoAA and CoAM), anti-CoAA (against CoAA only) and anti-active caspase-3 (against cleaved caspase-3) antibodies. Two representative views are shown (×400). Enlarged views are shown below. The results show CoAM and active caspase-3 staining in the EB cavity. () Western blot analysis of CoAA, CoAM and CoAR at indicated differentiation stages of P19 cells. () Immunohistochemistry analyses of mouse embryonic brain at gestation stages of E12.5 and E15.5. The sagittal sections were stained with affinity-purified anti-CoAA antibody (1:200) and counterstained with hematoxylin (×400)

Overexpression of CoAM and treatment with CoAA siRNA induce Sox6 expression in the absence of retinoic acid

<p><b>Copyright information:</b></p><p>Taken from "Switched alternative splicing of oncogene CoAA during embryonal carcinoma stem cell differentiation"</p><p></p><p>Nucleic Acids Research 2007;35(6):1919-1932.</p><p>Published online 1 Mar 2007</p><p>PMCID:PMC1874587.</p><p>© 2007 The Author(s)</p> P19 cells were transfected with CoAM or with CoAA-specific siRNA (25 nM) at the EC stage, and then were cultured in suspension for 4 days in the absence of RA induction. The cells were trypsinized, plated and further cultured for 3 days. The untreated and RA-induced (for 4 days) P19 cells were similarly cultured as controls. RT-PCR analyses were carried out with indicated marker genes at each indicated stage

Tentative model of CoAA alternative splicing regulation in the embryoid body of stem cells

<p><b>Copyright information:</b></p><p>Taken from "Switched alternative splicing of oncogene CoAA during embryonal carcinoma stem cell differentiation"</p><p></p><p>Nucleic Acids Research 2007;35(6):1919-1932.</p><p>Published online 1 Mar 2007</p><p>PMCID:PMC1874587.</p><p>© 2007 The Author(s)</p> CoAA is produced by alternative splicing with inclusion of the second exon. A competitive 5′ alternative splicing event excluding a portion of the second exon (2b) produces a dominant negative variant, CoAM, which lacks the activation domain. The basal promoter of the CoAA gene, shown as an open box, promotes splicing of the CoAA form, which is expressed in the outer layer of the embryoid body (gray). The upstream -regulating sequence, shown as a filled box, promotes alternative splicing of CoAM, which is expressed in the cavity of EB (white). The loss of upstream -regulating element in cancer may prevent the alternative splicing switch and disrupt stem cell differentiation

Alternative splicing switch from CoAA to CoAM during P19 embryonal carcinoma cell differentiation

<p><b>Copyright information:</b></p><p>Taken from "Switched alternative splicing of oncogene CoAA during embryonal carcinoma stem cell differentiation"</p><p></p><p>Nucleic Acids Research 2007;35(6):1919-1932.</p><p>Published online 1 Mar 2007</p><p>PMCID:PMC1874587.</p><p>© 2007 The Author(s)</p> () Undifferentiated P19 cells (EC) were induced with 500 nM retinoic acid (RA) in suspension culture up to 4 days to form embryoid bodies (EB2–EB4) which were trypsinized and further differentiated in the tissue culture dish for an additional 12 days in the absence of RA (D3–D12). Total RNA was isolated and normalized at each stage and analyzed using gene-specific primers as indicated by RT-PCR. GAPDH was the control. () CoAA and CoAM were analyzed by RT-PCR using shared primers. () The relative quantity of CoAA and CoAM was analyzed by quantitative real-time PCR. () Mouse ES cells were induced with 1 μM RA for 6 days. The EB was undisrupted in continuous culture for 15 days in the absence of RA. RT-PCR was carried out using RNA isolated at each stage