Figure 5: Cytological validation of SMC2 and CAP-H enrichment at telomere repeats.

(a) Telomere-FISH probes (green) and immunostaining for SMC2 or CAP-H SBP-tagged proteins (red) in both non-stretched and stretched mitotic chromosomes in chicken DT40 cells. DNA is co-stained with DAPI. The overlap of the FISH probe (telomere) and SBP signals (SMC2 or CAP-H) are indicated by a white arrow in both non-stretched (top two panels) and stretched chromosomes (bottom two panels). Their signal is also detected in the centromeric regions (primary constrictions of large chromosomes) in CAP-H–GFP–SBP cells (yellow arrow). (b) An example of chromosome telomere sequences colocalized with condensin I (CAP-H–GFP–SBP) on chromosome 1 that was used for quantification in (c). Note chromosome 1 has prominent interstitial telomere enrichment³⁶. Chromosomes were selected that had slightly stretched CAP-H–SBP (red) and therefore discontinuous signal, thereby minimizing the chance of random overlap. For scoring, all seven macro avian chromosomes from interstitial and canonical telomere-CAP-H overlapped regions were analysed, with both regions showing a very high overlap of CAP-H and telomere signal. The scale bar is 4 μm. (c) Quantification of telomere and CAP-H overlap in mitotic DT40 cells. Approximately 67% of telomere-FISH overlaps with SBP (CAP-H). This contrasts to 31% of SBP occupancy in DNA (representing the chance of SBP overlapping with random DNA). Telomere, CAP-H and DNA signals were calculated from chromosomes from 43 separate cells (n=43). The P-value of 1.29 × 10⁻⁷ is seen using Pearson’s χ² test comparing CAP-H and telomere overlap against CAP-H occupancy in the DNA (denoted by asterisk).