To identify optimal time points for transcriptomic analysis, we measured changes in the mRNA expression of phenotypic markers SOCS3 (23) and CD206 (12). As expected, IFN-γ but not IL-4 induced SOCS3 mRNA expression (Figure 1A), which was significant by 6 h and remained maximal at 18 and 32 h: it then declined but was still significantly elevated above untreated controls at 48 h (Figure 1A). Also as expected, IL-4 stimulated but IFN-γ inhibited CD206 mRNA expression after 48 h (Figure 1B). Interestingly, however, IFN-γ and IL-4 both induced CD206 to a similar extent at 6 h but the effect of IL-4 increased further from 18-48 h, whereas that of IFN-γ waned such that CD206 expression had declined below control levels after 32 and 48 h (Figure 1B). We, therefore, chose the 18 h and 48 h samples from these experiments to capture the differential effects of IFN-γ and IL-4 on steady-state mRNA levels of 84 epigenetic regulators using a commercially available RT-qPCR array. The combination of IFN-γ and IL-4 was also investigated because antagonistic interactions might enhance phenotypic differences (see Introduction). The results were normalized against a panel of five housekeeping genes integral to the profiler and significant changes were identified by using the array manufacturer’s software, which yielded values of mean fold change and p values after false discovery rate correction (Table 2). Eight significant differences and three non-significant trends that were subsequently found significant by conventional RT-qPCR are highlighted in bold. There were more significant responses to IFN-γ (10 gene changes at 18 h, of which 7 persisted at 48 h) than IL-4 (7 gene changes, only 2 of which were significant at both 18 h and 48 h). Contrary to our expectation of antagonistic responses, IFN-γ and IL-4 appeared to produce additive effects on the expression of epigenetic regulators (Table 2). The significant changes from the RT-qPCR screen were validated and extended by a detailed time course study using the full set of samples (Figures 2A–C and 3A–H). All the significant changes observed in the array were confirmed; and also the trends toward increase of KDM6B by IL-4 and decrease of KAT2A and SETD6 by IFN-γ were shown to be significant by standard RT-qPCR normalized against total RNA, which meets current recommendations (24). Only three genes, namely CIITA, KDM6B, and NCOA1 showed increased expression (Figures 2A–C), whereas the other 8, namely AURKB, ESCO2, KAT2A, PRMT7, SETD6, SMYD3, SUV39H1, and WHSC1 showed decreased expression (Figures 3A–H). The IFN-γ-induced increases in CIITA, KDM6B, and NCOA1 showed a similar pattern to SOCS3, with significant induction by 6 h and a tendency to decline thereafter (Figures 1A and 2A–C). The IL-4-induced increase in KDM6B was also significant by 6 h of treatment but then declined, unlike CD206, which remained elevated (Figures 1B and 2B). The decreased expression of AURKB, ESCO2, KAT2A, PRMT7, SETD6, SMYD3, SUV39H1, and WHSC1 was delayed to 18 h and beyond (Figures 3A–H). In the few cases, where it was possible, we sought to confirm the mRNA data by protein or activity measurements. In the case of SMYD3, IFN-γ significantly reduced protein levels after 48 h (Figure 4A). Either IFN-γ or IL-4 inhibited AURKB activity measured by the phosphorylation of histone3 on serine-10 (H3pS-10) (Figure 4B) (25). Consistent with this, we demonstrated that AZD1152, a pharmacological inhibitor of AURKB (26) also inhibited H3pS-10 to the same extent as either IFN-γ or IL-4 (Figure 4C).