Human pluripotent embryonal carcinoma NTERA2 cl.D1 cells maintain their typical morphology in an angiomyogenic medium

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Human pluripotent embryonal carcinoma NT2/D1 in an angiomyogenic medium. TEM images at differentiation day 20.

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Human pluripotent embryonal carcinoma NT2/D1 in an angiomyogenic medium. RT-PCR Qualitative analysis. This is not a quantitative analysis, what is the best choice to compare transcripts of different genes. However, a qualitative analysis can be done, comparing the intensity of the gel bands with the intensity of a house keeping gene, in our case the β actin. The scores we have chosen to construct these graphics were: 0 – PCR product not detected; 1 – PCR product with much less intensity of the β actin product; 2 – PCR product with less intensity of the β actin product; 3 – PCR product with the intensity of the β actin product; 4 – PCR product with more intensity of the β actin product; 5 – PCR product with much more intensity of the β actin product. Of course, that with this method the conclusions one can take are very limited, and only big differences have caught our attention. The main observations were: i) contrarily to what we have expected, in general, all genes were equally expressed in the different time points analyzed, with the exception of the MYH7 that was never detected; ii) the gel band of CRIPTO has tendency to change to a specific pattern during NT2/D1 differentiation; iii) a DNA band of BMPRIB appears in late stages of NT2/D1 differentiation. We cannot exclude DNA contamination in the PCR reaction, but if so, and because all the other reactions used the same cDNA, why was this gene the only one presenting a DNA amplified band? We do not have a explanation for this. iv) the heavy band of BMPRII appears only in late stages of NT2/D1 differentiation.

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Human pluripotent embryonal carcinoma NT2/D1 in an angiomyogenic medium. The case of the MYH7 PCR. The MYH7 primers were constructed and positively verified for accuracy using cDNA samples of human masseter and human left ventricle, which are muscle tissues known to express this gene in a high relative level. With that samples we could obtain single band PCRs positively verified by sequencing. (see the two last lanes before the ladder in the gel image). The first time we have used the MHY7 primers in non muscle tissues, we have obtained a pattern of expression that was very interesting at the beginning, because we thought that we were leading with an alternative splicing event. However, after sequencing, the PCR products of different sizes that the NT2/D1 and other samples gave rise in the polymerase reaction with the MYH7 primers, had nothing to do with the expected results, as one can see in the figure. The five non specific bands in the NT2/D1 lane are marked from 1 to 5 with the respective sequentiation results shown below.

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Human pluripotent embryonal carcinoma NT2/D1 in an angiomyogenic medium. 100× images. Phase contrast, hystochemical and fluorescent images of NT2/D1 cells at time points 1, 5, 10, 20 and 30 days in Control, IAM and IAM+BMP2 culture mediums. Transcriptional analysis of 16 genes at the same time points is also shown.

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Diagram of the NT2/D1 Transcripts

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Primer data and sizes of the PCR amplified products

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