Enhanced sister chromatid cohesion is a distinctive feature of the active VSG ES. Different cell lines with GFP-tagged loci described previously (Landeira and Navarro, 2007) were subjected to indirect IF using polyclonal anti-GFP antibodies and DAPI staining. (A) Histogram showing the percentage of nuclei containing two GFP dots in 2K1N bloodstream form trypanosomes with a GFP tag in either the rDNA locus (n = 196), VSG-BC (n = 166), inactive VSG ES (n = 104), or active VSG ES (n = 161). (B) Histogram showing the percentages of cells containing two GFP dots in 2K1N procyclic form trypanosomes with a GFP tag in either the rDNA locus (n = 166), silenced VSG ES (n = 184), or procyclin locus (n = 170). (A and B) Dashed lines indicate the value of the rDNA that was used as the expected value in Fisher's exact test. (C and D) Representative pictures of a 1K1N (C) or 2K1N (D) cell showing GFP dot detection in a double GFP-tagged bloodstream cell line, with GFP marking the rDNA locus (250 lac operator repeats; arrows) and active VSG ES (35 lac operator repeats; arrowheads). (E–M) 2K1N bloodstream form cell lines tagged with GFP in the rDNA locus (n = 170), inactive VSG ES (n = 201), or active ES (n = 186) were grouped based on DAPI staining (Fig. S2) as G2 prophase, metaphase, or anaphase and scored as containing either one or two GFP dots. Representative pictures show GFP dot detection (arrowheads) in cell lines GFP tagged in the rDNA locus (E–G), inactive VSG ES (H–J), and active VSG ES (K–M). Insets show DAPI signal distribution by applying a gradient LUT (see Materials and methods), for which red corresponds to lower intensity pixel values and green to higher values. (C–M) Asterisks mark the kinetoplast. Bars, 1 µm.