Materials and Methods s20

Supplemental Data

Supplemental Figures

Figure S1, related to Figure 1. (A) Knockdown efficiency of various siRNAs was measured by RT-PCR. (B) HeLa cells transfected with control, EHMT1a or EHTM1b siRNAs were treated with or without TNFa for 2 hours, and the expression of IL8 and EHMT1 were measured by RT-PCR. (a-b) Error bars represent the variation range of duplicate experiments. (C) HeLa cells transfected with control or EHMT1 siRNAs were treated with or without IL1b for 2 hours, and the expression of A20, IP10 and IL8 were measured by RT-PCR.

Figure S2, related to Figure 1. (A) HeLa cells transfected with control or EHMT1-specific siRNAs were stimulated with or without TNFa for the indicated time periods, and cytoplasmic or nuclear extracts were immunoblotted with the indicated antibodies. Ns: non-specific. (B) Whole cell extracts from HeLa cells treated as in (A) were immunoblotted with the indicated antibodies.

Figure S3, related to Figure 4. (A) HeLa cells were pretreated with or without (medium) Bix for 24 hours and then stimulated with TNF for 4 hours before addition of ActD. The mRNAs were measured by RT-PCR. (B) MEF cells were analyzed by ChIP (left panel, IgG and p65, *: p<0.05). MEF cells treated with or without TNF for 30 minutes were analyzed by p65 ChIP (right panel, *: p<0.05). (C) HeLa cells transfected with or without p50-specific siRNA were analyzed by ChIP (histone H3, H3K9me2 and EHMT1; mean±SD, data are representative of two independent experiments, *: p<0.05). (D) HeLa cells transfected with control or EHMT1-specific siRNAs were treated with or without TSA (200nM) for 18hr followed by 2 hours TNFa stimulation. The expression of A20, IL8, TNFa, IL6 and IFNb were measured by RT-PCR. (E) HeLa cells transfected with or without EHMT1-specific siRNA were analyzed by ChIP (HDAC1; mean±SD, data are representative of two independent experiments).

Figure S4, related to Figure 5. (A) HeLa cells were treated with 5uM BIX01294 or DMSO for 48 hours and then infected with increasing MOI of VSV-GFP for 24 hours. Cells that were not killed by the virus were stained with amido black. (B) Whole cell extracts were prepared from A549-PB1 cells were treated with 5mM BIX01294 or DMSO for 48 hours, and immunoblotted with the indicated antibodies.

Table S1, related to Figure 1c. List of genes induced by TNFa in control and EHMT1 siRNAs transfected HeLa cells. Values represent the fold of induction.

Table S2, related to Figure 1, 2, 4 and 5. Sequences of primers used for RT-PCR and ChIP assays.

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