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contains cell culture methods and Supplemental Figure 1. Four pair-wise comparisons of marker gene expression in cell lines. The most highly expressed markers that differ between two lines are labeled using a human symbol of a homologous gene

The diploid anuran, Xenopus tropicalis, has emerged as a key research model in cell and developmental biology. To enhance the usefulness of this species, we developed methods for generating immortal cell lines from Nigerian strain (NXR_1018, RRID:SCR_013731) X. tropicalis embryos. We generated 14 cell lines that were propagated for several months. We selected four morphologically distinct lines, XTN-6, XTN-8, XTN-10 and XTN-12 for further characterization. Karyotype analysis revealed that three of the lines, XTN-8, XTN-10 and XTN-12 were primarily diploid. XTN-6 cultures showed a consistent mixed population of diploid cells, cells with chromosome 8 trisomy, and cells containing a tetraploid content of chromosomes. The lines were propagated using conventional culture methods as adherent cultures at 30 °C in a simple, diluted L-15 medium containing fetal bovine serum without use of a high CO2 incubator. Transcriptome analysis indicated that the four lines were distinct lineages. These methods will be useful in the generation of cell lines from normal and mutant strains of X. tropicalis as well as other species of Xenopus.

Additional file 1: Figure S1. (a) The graphics in abdominal plain CT and (b) the CECT equilibrium phase of case1 are shown. (c) The chest CT of case1 also revealed multiple lung nodules (arrow). (d) The lesions in the liver of case1 exhibited high signal intensity on axial T2WI. (e) The arterial dominant phase and (f) the hepatocellular phase in Gd-EOB-DTPA are shown. (g) In immunostaining (200×), a histological section of a hepatic specimen obtained via percutaneous liver needle biopsy in case 1 was positive for factor VIII. Figure S2. (a) The noncontrast CT of case2 revealed multiple hypodense liver nodules, with cystic lesions. (b) In the equilibrium phase of CECT, enhancement persisted inside the tumors. (c) T2WI showed multiple hyperintense liver nodules. (d) The size of liver tumors in the arterial dominant phase of CECT and (e) in the equilibrium phase have not increased when approximately 70 months has passes after the diagnosis was made. (f) In immunostaining (200×), a histological section of a hepatic specimen obtained via percutaneous liver needle biopsy in case 2 was positive for factor VIII. Figure S3. (a) The noncontrast CT of case3 revealed multiple hypodense liver nodules. (b) The graphics of abdominal CT in the arterial dominant phase and (c) in the equilibrium phase are shown. (d) The arterial dominant phase in Gd-EOB-DTPA is shown. (e) The size of liver tumors of case3 in the arterial dominant phase of CECT have not increased when approximately 49 months has passes after the diagnosis was made. (f) In immunostaining (200×), a histological section of a hepatic specimen obtained via percutaneous liver needle biopsy in case 3 was positive for factor VIII.

Extracellular factors belonging to the TGF-β family play pivotal roles in the formation and patterning of germ layers during early Xenopus embryogenesis. Here, we show that the vg1 and nodal3 genes of Xenopus laevis are present in gene clusters on chromosomes XLA1L and XLA3L, respectively, and that both gene clusters have been completely lost from the syntenic S chromosome regions. The presence of gene clusters and chromosome-specific gene loss were confirmed by cDNA FISH analyses. Sequence and expression analyses revealed that paralogous genes in the vg1 and nodal3 clusters on the L chromosomes were also altered compared to their Xenopus tropicalis orthologs. X. laevis vg1 and nodal3 paralogs have potentially become pseudogenes or sub-functionalized genes and are expressed at different levels. As X. tropicalis has a single vg1 gene on chromosome XTR1, the ancestral vg1 gene in X. laevis appears to have been expanded on XLA1L. Of note, two reported vg1 genes, vg1(S20) and vg1(P20), reside in the cluster on XLA1L. The nodal3 gene cluster is also present on X. tropicalis chromosome XTR3, but phylogenetic analysis indicates that nodal3 genes in X. laevis and X. tropicalis were independently expanded and/or evolved in concert within each cluster by gene conversion. These findings provide insights into the function and molecular evolution of TGF-β family genes in response to allotetraploidization.