Figure 4. Partial knockdown of D2HGDH in B-cell lymphoma cell lines significantly modifies histone/DNA methylation and HIF1α hydroxylation.
(a) SiRNA-mediated partial knockdown of D2HGDH with two targeting oligonucleotides increased the methylation levels of H3K4me3 in comparison with cells transfected with a control siRNA. (b) Under hypoxia (1% O2, 16 h), B lymphoma cells with partial suppression of D2HGDH expression displayed lower HIF1α hydroxylation and consequent stabilization of total HIF1α. In a and b, the extent of D2HGDH suppression is shown by western blotting and densitometry quantifies all relevant changes. (c) The levels of 5hmC and 5mC (top and bottom panels) were significantly lower and higher, respectively, in cells with a D2HGDH KD when compared with their isogenic controls (P<0.0001, ANOVA, P<0.05 Bonferroni's multiple comparison test for si-ctrl versus si-D2#3 or si-D2#5). The 5hmC and 5mC measurements shown are the mean and s.d. of three data points derived from three biological replicates. The transient knockdown of D2HGDH in these cell lines was repeated three times.