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. 2019 Jul 3;218(8):2492–2513. doi: 10.1083/jcb.201810166

Figure 7.

Figure 7.

Tetraploid derivatives of human hTERT CHON-002 cell line inactivate some rDNA loci. These UBF-negative rDNA loci are not incorporated in nucleoli and do not form rDNA linkages. (A) Quantification of total number of rDNA loci and active (UBF+) number of rDNA loci in RPE1 and CHON cell lines. Lines between data points connect total and UBF+ rDNA loci in the same chromosomal spread. In RPE1, all rDNA loci were UBF positive in all cells. In most parental CHON-002 cells, all rDNA loci were UBF positive, with a few cells silencing one rDNA locus. In tetraploid CHON derivatives, all cells had some silenced rDNA loci. 10 spreads per cell line were analyzed. (B) Ploidy analysis of asynchronously growing tetraploid single-cell clones, designated CHON tetraploid-1 and CHON tetraploid-2, generated from hTERT CHON-002 cell line. Cells were fixed and stained with propidium iodide. FACS profiles of CHON tetraploid-1 and CHON tetraploid-2 indicate the doubling of the DNA content. (C) Chromosome spreads from diploid CHON-002 and tetraploid CHON tetraploid-1 cells were labeled by immuno-FISH with rDNA probe (green) and UBF antibody (red). Boxes A–C highlight rDNA linkages that were formed between UBF+ rDNA loci (magnified on the right; bar, 1 µm). Arrows 1–3 point to rDNA chromosomes lacking UBF. Panels below show individual rDNA chromosomes labeled with rDNA probe (top row) and UBF antibody (bottom row). In the spread from the parental CHON-002 cell line, there were 10 rDNA chromosomes, all UBF positive. In the spread from the CHON tetraploid-1 derivative, there were 19 rDNA chromosomes, 16 UBF+ and 3 UBF− (boxes 1–3, magnified on the right). All rDNA linkages were formed between UBF-positive rDNA chromosomes. Bar, 10 µm. (D) Interphase nuclei from diploid CHON-002 (top) and tetraploid CHON tetraploid-1 (bottom) cells were labeled by immuno-FISH with rDNA probe (green), nucleolin antibody (red), and UBF antibody (magenta). Bar, 10 µm. In the parental CHON-002 cell nucleus, all rDNA was decompacted and associated with UBF and nucleolin. In the tetraploid derivative nucleus, compact rDNA loci were present (arrows) that were UBF negative and were not incorporated in nucleoli (magnified insets; bar, 1 µm).