Quantification of Global H3K9 Methylation

To compare levels of global H3K9 methylation of samples at different developmental stages and between untreated and BIX-treated samples, an EpiQuik Global Histone H3K9 Methylation Assay Kit (Colorimetric) (EpiGentek, Farmingdale, NY, United States) was used according to the manufacturer’s instruction. Hundred milligram of each sample were homogenized, nuclei and histone extraction were sequentially performed following the extraction procedure and solutions of the kit. Histone protein concentration was measured by the Bradford method, and the protein concentrations of histone extracts were adjusted in all samples to 300 ng/μl. In short, 1.5 μg histone proteins were spotted on the strip wells. Methylated histone H3K9 was detected with a specific antibody which was bound to a horseradish peroxidase-conjugated secondary antibody; amounts of methylated histone H3K9 were quantified by a color development reagent and were proportional to the intensity of color. Color density was measured by absorbance (optical density, OD) on the microplate reader at 450 nm and the amount of methylated H3K9 was proportional to the OD. Blanks and negative control (standard histone extract with no H3K9 methylation, provided in the assay kit) OD readings were subtracted to the sample OD readings. Assays were performed in triplicate. Results are presented as mean OD ± SE. Significant differences were tested by one-way ANOVA analysis of variance followed by Tukey’s multiple comparison test at P ≤ 0.05.