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. 2011 Dec 5;6(12):e28509. doi: 10.1371/journal.pone.0028509

Figure 4. Schematic showing STAT6 silencing effects on cholesterol biosynthesis and validation of FOXJ2 binding by EMSA.

Figure 4

a) siRNA mediated STAT6 silencing increases the cholesterol levels of the cell by enhancing the expression levels of enzymes involved in cholesterol biosynthesis pathway. # represents the rate limiting step of the cholesterol biosynthesis pathway. b) The binding sites of FOXJ2 and FOXD3 to the 5kB region upstream of transcription start site of these genes as revealed by OTFBS. 0 bp marks the transcription start site of the gene. c) The transcription factors common to the 3 enzymes of cholesterol biosynthesis pathway that got upregulated after STAT6 silencing were predicted by OTFBS. * transcription factor binding validated by EMSA. 18 μg of nuclear extracts from untransfected or siRNA transfected NCI-H460 cells were incubated with wild type labeled oligonucleotide containing the binding elements of FOXJ2. Mutated probe was used to check the specificity of the DNA-protein complexes. On termination of incubation, samples were resolved in a non-denaturing polyarylamide gel and subjected to phosphorimager analysis. Lane 1: labeled FOXJ2 specific oligos, lane 2: untransfected NCI-H460 cells incubated with labeled FOXJ2 specific oligos, lane 3: NCI-H460 cells tranfected with STAT6 specific siRNA at 48 h incubated with labeled FOXJ2 specific oligos, lane 4: NCI-H460 cells tranfected with STAT6 specific siRNA at 72 h incubated with labeled FOXJ2 specific oligos, lane 5: NCI-H460 cells tranfected with scrambled siRNA incubated with labeled FOXJ2 specific oligos, lane 6: untransfected NCI-H460 cells incubated with labeled mutated FOXJ2 specific oligos.