FIG. 10.

Methyltransferase enzyme inhibition potentiates the ability of ascorbic acid to promote the DN-to-DP transition in OP9-DL1 cultures. (A) OP9-DL1 cocultures were maintained without ascorbic acid for 19 days, at which time RG108 (5 μM) or BIX01294 (50 nM) was added with or without 100 μM ascorbic acid. Cultures were evaluated 8 days later as described in the legend for Figure 9. Values from replicate 1 ml cultures are shown with mean±standard error. (B) Cultures maintained without ascorbic acid for 19 days were replated in the presence or absence of 30 nM BIX01294 and cultured for an additional 8 days. Lymphocytes obtained from these cultures were processed for chromatin immunoprecipitation (ChIP) using antibodies as indicated. The fold enrichment of the Cd8a promoter region relative to β-actin DNA is shown as the mean±standard error of triplicate technical replicates. (C) Cultures maintained without ascorbic acid for 21 days were replated with or without ascorbic acid and harvested for ChIP 72 h later. ChIP for total histone H3 protein was performed and analyzed as in Panel (B). Data indicate mean±standard deviation of 2 (no ascorbic acid) or 3 (+ascorbic acid) biological replicates with three technical replicates each.