Figure 2

Applying the AZA-MS method to study an in vitro AZA-treated cell line. (a) Two-factor experiment to determine minimum input amount of DNA required to reliably detect DNA-5-AZA-CdR. Signal intensities of DNA-5-AZA-CdR measured from different input quantities of DNA (0–1250 ng, x axis) extracted from RKO cells treated with different concentrations of AZA (100−1250 nM) for 3 consecutive days. A quantity of 500 ng (highlighted with a shaded box) was determined as the minimum amount of DNA to reproducibly detect good signal across all the tested AZA treatment dosages. Points represent mean of three independent experiments, and whiskers represent s.d. (b) Experiment to determine minimum input amount of RNA required to reliably detect RNA-incorporated AZA. Signal intensities of RNA-incorporated AZA measured from different input quantities of RNA (0–1250 ng, x axis) extracted from RKO cells treated with different concentrations of AZA (100−1250 nM) for 3 consecutive days. A quantity of 500 ng (highlighted with a shaded box) was determined as the minimum amount of RNA to reproducibly detect good signal across all the tested AZA treatment dosages. Points represent mean of three independent experiments, and whiskers represent s.d. (c) Schematic of the AZA-MS assay, illustrating the separation of the various subcellular components (cytoplasmic nucleotides, RNA and DNA) from the same sample before LC–MS. (d) Allelic bisulphite sequencing of the MLH1 locus in RKO cells. Top panel shows the schematic of the gene, with a CpG island (green box) and the region assayed for CpG methylation (black box). Lollipop visualisations of the methylation status in control, DMSO treated cells (‘untreated’, left panel) and RKO cells treated for 3 days 1.25 μM AZA (right panel) are shown. Equivalent bar-graph quantification of the data is presented on the right. Whiskers represent s.d. ** P-value <0.05, Student’s t-test. (e) Quantitative real-time PCR (qRT-PCR) of MLH1 expression levels in RKO cells treated with different doses of AZA (0–1250 nM) for 3 days, showing robust re-expression after treatment with 1250 nM AZA. (f) AZA quantification in cytoplasm and RNA from RKO cells, treated either with 1.25 μM of AZA (red graphs) or control (DMSO, blue graphs) for 3 days. The calibration curve used for AZA quantification is shown, along with AZA chemical structure and the R² value. Abundance measurements for AZA in the cytoplasm (left graph) and incorporated into RNA (right graph) are shown. Data are from triplicate experiments, with s.d. depicted by whiskers. ***P-value <0.001, Student’s t-test. (g) 5-AZA-CdR quantification in cytoplasm and RNA from RKO cells, treated either with 1.25 μM of AZA (red graphs) or control (DMSO, blue graphs) for 3 days. The calibration curve used for 5-AZA-CdR quantification is shown, along with 5-AZA-CdR chemical structure and the R² value. Abundance measurements for 5-AZA-CdR in the cytoplasm (left graph) and incorporated into DNA (right graph) are shown. Data are from triplicate experiments, with s.d. depicted by whiskers. ***P-value <0.001, Student’s t-test.