Fig 1: CENP-A is overexpressed in colon cancer SW480 cells, and associates with H3, ATRX, and DAXX. (A) Upper panel: Western blot (WB) analysis of total nuclear CENP-A, HJURP, ATRX, and DAXX, relative to core histone H4 across cell lines. Lower panel: quantification of CENP-A protein expression standardized to normal colon (replicate data from Table 1), error bars = SEM. (B) Upper panel: semi-quantitative PCR analysis of CENP-A mRNA expression in normal and SW480 cells in three replicates. Lower panel: quantification of the CENP-A mRNA expression normalized to β-actin from four experiments, error bars = SEM. (C) Scheme depicting strategy to separate centromeric from ectopic CENP-A nucleosomes and subsequent experiments performed with each fraction. (D) Micrococcal nuclease (MNase) ladders of input chromatin used subsequently in the primary CENP-B chromatin immunoprecipitation (ChIP) for normal, HeLa and SW480 cell lines demonstrates that mostly mono-, di-, and tri-nucleosomal arrays are present in the input chromatin. Slight variations in digestibility derive from intrinsic variability in the different cell lines’ biology. (E) WB slice panels show CENP-B, CENP-A, H2A.Z, and H3 protein analysis from CENP-B IP and sequential CENP-A IP from normal, HeLa, and SW480 cells. Mock immunoprecipitation (IP) indicates control immunoprecipitation with nonspecific IgG. Recombinant CENP-A (Rec. CpA) was used as a detection specificity control. Gray CENP-A arrow in third row indicates a band that was already present prior to H2A.Z probing. Data quantification is provided in Table 2. (F) WB for ATRX and DAXX in normal, HeLa, and SW480 CENP-B IPs versus ectopic CENP-A IPs (data summarized in Table 2).