Matin, they might play different roles in terms of the surrounding centromeric chromatin.Table 1 Analysis of total chromosome number in each cell after 24 and 48 h of trichostatin A (TSA) treatmentWI-38 Control Mode Loss Gain 4n Total 42 (87.5 ) 6 (12.5 ) 0 0 48 24 h* 21(42 ) 8 (16 ) 5 (10 ) 16 (32 ) 50 48 h*,** 28 (54.9 ) 11 (21.56 ) 2 (3.92 ) 10 (19.6 ) 51 HCT116 Control 49 (96.08 ) 2 (3.92 ) 0 0 51 24 h 25 (54.9 )* 14 (27.5 )* 10 (19.6 )* 2 (3.9 ) 51 48 h** 40 (78.43 ) 7 (13.73 ) 4 (7.84 ) 0Loss was considered below 2n, and gain was considered above 2n. *Levene’s test p < 0.001; treatment versus control. **Levene's test p < 0.05; 24-h treatment versus 48-h treatment.Gonz ez-Barrios et al. Cell Division (2014) 9:Page 4 ofFigure 2 Centromeric localization of HP1 and HP1 in WI-38 cells under basal conditions and after TSA treatment. (A) WI-38 cell fluorescent microscopy localization of CENP-A with H3K9me3 (lane 1), HP1 (lane 2-3) and HP1 (lane 4). (B) Chromatin localization by fluorescent microscopy in WI-38 cells after TSA treatment comparing H3K9me3 with HP1 (lane 1) or HP1 (lane 2) H3K9ac with HP1 (lane 3) or HP1 (lane 4), the centromeric localization of HP1 compared with CENP-A (lane 5), and HP1 compared with ACA (lane 6). The DNA is marked with DAPI; the images show PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26024392 the most common distribution of proteins after the analysis of 100 cells ( ); the boxes represent a magnification of the immunofluorescence results; M, mitotic cell.We then determined whether the dynamics and localization of H3K9me3, HP1, and HP1 are conserved in a cancer cell line with no CIN and with stablechromosome segregation such as HCT116 cells. For this purpose, we performed immunofluorescence microscopy assays to observe the centromeric localization of HP1.Gonz ez-Barrios et al. Cell Division (2014) 9:Page 5 ofFigure 3 Centromeric localization of histone marks, HP1, and HP1 in HCT116 cells under basal conditions and after TSA treatment. (A) HCT116 chromatin localization by fluorescent microscopy of CENP-A with H3K9me3 (lane 1) or HP1 (lane 2), H3K9me3 and HP1 (lane 3), ACA and HP1 (lane 4), and HP1 and Mis12 (lane 5). (B) Centromeric localization of H3K9me3 and CENP-A (lane 1), CENP-A and HP1 (lane 2), ACA and HP1 (lane 3) and Mis12 Tirabrutinib web co-localization with HP1 (lane 4). The DNA is marked with DAPI; the images show the most common distribution of the proteins after the analysis of 100 cells ( ); the boxes represent a magnification of the immunofluorescent results; M, mitotic cell.H3K9me3 co-localized with CENP-A during interphase; during mitosis, it either co-localized or was enriched in the region neighboring the centromere (Figure 3A). As expected, the HP1 isoforms showed similar patterns of localization in both cell lines, suggesting conserved chromatin behavior for both H3K9me3 and HP1 (Figure 3A). We observed almost no co-localization of the euchromaticmarks H3K9ac and H3K4me2 with CENP-A, indicating that open chromatin marks are present at centromeric regions at a low frequency in both cell lines (Additional file 1: Figure S1A-B). Considering that HP1 has been associated with the Mis12 complex, we observed that HP1 colocalizes with Mis12 during interphase, but this localization changes slightly during mitosis (Figure 3A).Gonz ez-Barrios et al. Cell Division (2014) 9:Page 6 ofTo confirm the presence of HP1 at centromeric chromatin and to assess its dynamics during interphase and mitosis, we selected HCT116 cells with stable chromosomal segregation. We treated.