The same samples as in Figure 6 were analyzed in this experiment. including two new rabbit polyclonal antibodies, generated against TERF1-tsi specific peptides, indicate nuclear localization of TERF1-tsi in a subset of spermatogonial stem cells. In line with this observation, immunofluorescence analyzes in various cell lines consistently revealed that ectopic TERF1-tsi localizes to the cell nucleus, mainly but not exclusively at telomeres. In a first attempt to evaluate the impact of TERF1-tsi in the testis, we have tested its expression in normal testis samples versus matched tumor samples from your same patients. Both RT-PCR and IHC show a specific downregulation of TERF1-tsi in tumor samples while the expression of TERF1 and PIN2 remains unchanged. genomic locus. Not drawn to level. E1CE11 symbolize the exons, including exon 9 (reddish) which is found in the new isoform, explained in this manuscript. E9 and E7 (green) are absent in the PIN2 splice variant while TERF1 lacks E9 only. The colored arrows indicate the location of the primer (blue, forward primer and reddish, reverse primer) which were used to amplify the three splice variants in a single PCR reaction (hmsFor and hmsRev, respectively). Open in a separate window Physique 2 Semi-quantitative PCR analysis showing the splice variants (top) and products (bottom). White arrow indicates the additional PCR product in the testis sample. PCR reaction was Rabbit polyclonal to ZBTB49 performed using the primer pair indicated in Physique 1. Of notice, we observed variations in TERF1 and PIN2 splice variants among the tissues (e.g., absence of TERF1 in the belly or absence of PIN2 in the lung tissue). Please also note that mRNAs are not visible in the offered figure due to low signal intensity, although both splice variants are expressed in these tissues. Open in a separate window Physique 3 Semi-quantitative PCR showing expression of the splice variants in human and mouse testes and human cell lines. PCR reaction was performed using the primer pair indicated in Physique 1. Of notice, we observed higher mRNA levels in the human cell lines in comparison to was not detectable in these cell lines. Also, note that the mouse is usually shorter compared to human encompasses an evolutionarily conserved novel exon, exon 9. (Top) Schematic Poziotinib drawing of the intron-exon structure of genomic locus. Not drawn to level. Exons 7 (green) and 9 (reddish) are color-indicated by the reddish rectangle. The lower-case x in the red rectangle (splice variants. The N-terminal acidic domain name, the dimerization domain name, and the DNA-binding domain name (DBD) are shown. In addition, exon 7 (E7), which is usually missing in PIN2, and exon 9 Poziotinib (E9), which is only present in TERF1-tsi, are indicated by a green or a reddish rectangle, respectively. Open in a separate windows Physique 5 TERF1-tsi expression is usually observed in human and chimpanzee testis samples. Results from the semi-quantitative PCR showing the expression in testis samples from (M.m.), (C.j.), (P.t.) and (H.s.). Please note that testis samples were not available from and specific primer pairs, primer pair A (left) and primer pair B (right), respectively. Plasmid DNA with cloned cDNA as well as vacant vector (EV), were used to control the specificity of the primer. Due to high plasmid DNA concentrations utilized for the positive control PCR reactions, poor, non-specific PCR products were also visible with the TERF1 cDNA. White arrows show expression in human (H.s.) and chimpanzee (P.t) testis samples. We used a set of commercially available RNAs extracted from 21 human tissue samples to detect splice variants, along with the loading control in a semi-quantitative RT-PCR reaction. As expected, we detected splice variants in all tissues analyzed here, although to varying levels. Interestingly, we observed an additional PCR product in the human testis sample, which was larger than both and (Figure 2, indicated with white arrow). To exclude potential PCR artefacts, next, we performed the same RT-PCR reaction using cytoplasmic RNA purified from two independent human testis samples along total RNA prepared from cultured human cells and with mouse testes RNA prepared from 5 different mice (Figure 3). Again, we observed three different PCR products with human testis RNA samples, corresponding to and an additional product, potentially corresponding to a novel splice variant. Of note, although expected products could be detected in mouse testis samples and the human cell lines, no additional band was Poziotinib observed in these.
