Figure 2. Methionine restriction alters the host and EBV epigenetic landscape.
A) 5mC dot blot analysis of DNA extracted from P3HR-1 cultured in 100 vs 10 μM Met for 72h. As a loading control, membranes were stained with ethidium bromide.
B) Immunoblot analysis of WCL from P3HR-1 cells cultured in cultured in 100 vs 10 μM Met for 72h.
C) Volcano plot of host epigenetic factor RNAseq from P3HR-1 (left) or Rael (right) cells cultured in 100 vs 10 μM Met for 72h, using the curated EpiFactors database (Medvedeva et al., 2015). P-value and Log2 fold change data were generated from n=2 replicates (P3HR-1) or n=3 replicates Rael by DESeq2, using default settings with Wald test and normal shrinkage, respectively.
D) ChIP-qPCR analysis of IgG control versus anti-UHRF1 abundances in chromatin extracted from P3HR-1 cells cultured in 100 vs 10 μM Met for 72h. Shown are C promoter (Cp) qPCR mean ± SD values from n=3 replicates. P values were calculated by 2-way ANOVA.
E-F) Immunoblot analysis of WCL from P3HR-1 or Rael cultured in 100 vs 10 μM Met for 72h.
G) Immunoblot analysis of puromycin incorporation into newly synthesized polypeptides (top) or of the indicated proteins from WCL prepared from the indicated B cell lines cultured in 100 vs 10 μM Met for 72h.
H) 5mC MeDIP analysis of chromatin from P3HR-1 cultured in 100 vs 10 μM Met for 72h. Shown are mean ± SD values from n=3 replicates of qPCR analysis of Cp, the LMP1 promoter (LMPp), the BZLF1 promoter (BZLF1p) or the BMRF1 promoter (BMRF1p). Cells were maintained in acyclovir (100μg/ml) to prevent lytic DNA synthesis. P values were calculated by 2-way ANOVA.
I) ChIP for H3K9me3 or H3K27me3 of chromatin from P3HR-1 cells cultured in 100 vs 10 μM Met for 72h, followed by qPCR with primers specific for the C, LMP1, BZLF1 or BMRF1 promoters. Cells were maintained in acyclovir (100μg/ml) to prevent unchromatinized lytic DNA synthesis. Shown are mean ± SD values from n=3 replicates. P values were calculated by 2-way ANOVA.
All blots are representative of n=3 replicates. See also Figure S3.