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. 2011 Apr 19;6(4):e18844. doi: 10.1371/journal.pone.0018844

Figure 5. Epigenetic changes after SETD2 knockdown.

Figure 5

A. Western blot for HBEC cells after non-targeting siRNA and SETD2 siRNA transfection using anti-H3K36me3 antibodies and unmodified histone H3 antibodies. B. Composite profile of epigenetic changes after SETD2 siRNA knockdown in HBEC. The profile was created for gene bodies marked by H3K36me3 and DNA methylation over at least 20% of gene body length. Each gene body was divided into 20 bins and the 5 kb upstream of the TSS and 5 kb downstream of the 3' gene end were divided into 10 bins. The average signal for each single bin is indicated. C. Analysis of DNA methylation in the gene body of the NOTCH3 gene in conditions of H3K36me3 deficiency. The H3K36me3 profile of NOTCH3 after non-targeting siRNA and SETD2 siRNA transfections in HBEC cells is shown. The region analyzed by COBRA methylation assays is indicated by a box. Using gene-specific primers, bisulfite-converted DNA was amplified. After cutting with HpyCH4IV recognizing CpG dinucleotides, mock (-) and enzyme-digested (+) PCR products were fractionated on a 2% agarose gel. In vitro CpG-methylated human DNA (M) served as a positive control. Cleavage indicates DNA methylation. D. Analysis of H3K36me3 and DNA methylation in the gene body of RFX2 in conditions of H3K36me3 deficiency. The region analyzed by bisulfite sequencing is marked by a box. Using gene-specific primers, bisulfite-converted DNA was amplified, cloned and 20 individual clones were sequenced. White circles, unmethylated CpG sequences; black circles, methylated CpG sequences. E. Western blot for HCT116-DKO cells after non-targeting siRNA and SETD2 siRNA transfection using anti-H3K36me3 antibodies and unmodified histone H3 antibodies. F. DNA methylation analysis of the gene body of NOTCH3 after non-targeting siRNA and SETD2 siRNA transfection of HCT116-DKO cells. Using gene-specific primers, bisulfite-converted DNA was amplified, cloned and 13 individual clones were sequenced. White circles, unmethylated CpG sequences; black circles, methylated CpG sequences.