Research Paper Volume 4, Issue 11 pp 823—842

Figure 3. Histone H2A variants and their transcription in HCA2 cells. (A) Alignment of all H2A variant protein sequences. Quality track scores the level of sequence conservation between all variants for each amino acid. Blue boxes highlight tryptic histone H2A family internal monitoring peptides used in SRM analysis. Red boxes highlight tryptic H2A.X-specific peptides. (B) RNA was prepared from untreated proliferating and bleomycin-treated HCA2 cells (acute DDR (+Bleo, 3 h) and drug-evoked senescence (+Bleo, 5 days)). RT-PCR analysis was used to quantify the mRNA abundance of a selection of H2A variants in each condition. H2A type 1 represents a number of H2A variants from the type 1 cluster (see A). The p-values for H2A.J and H2A type 1 are shown, calculated using Student's t-test, two-tailed, unequal variance, between the conditions. Each condition consists of biological triplicates, with further technical triplicates for each. Histone H2A family members can be subdivided into low expression (H2A type 3, H2A.Z, macro-H2A.1, H2A.V), medium expression (H2A.J), and high expression (H2A type 1), based on their expression relative to β-actin.