Fig. 6

TRIM28 negatively regulates necrosome-induced cytokine production. (A) mRNA expression levels of HT-29 cells expressing TRIM28 shRNA or the non-silencing control were analyzed by qPCR. (B) Cells from (A) were treated with TSZ for the indicated time points and analyzed by western blotting (left panel), LDH leakage (middle), and MTT assay (right panel). (C and D) Cells were treated with TSZ for indicated times. Their mRNA levels were analyzed by qPCR (C), and cell lysates were subjected to western blotting (D). (E and F) SNU-620 cells stably expressing TRIM28 shRNA or RIPK3 shRNA were treated with TSZ in a time-dependent manner. Cell lysates were analyzed by western blotting (E), and mRNA levels were analyzed by qPCR (F). (G) L929 cells were treated with TZ for 2 h, and IL-6 and CCL4 mRNA levels were analyzed by qPCR. (H) HT-29 cells expressing TRIM28 shRNA or a non-silencing control were fractionated into nuclear and cytosol fractions after the indicated treatments. Fractionation samples were analyzed by western blotting. (I) Representation of motifs enriched at TSZ-sensitive TRIM28 loss peaks (top) and TSZ-sensitive TRIM28 gain peaks (bottom). Known motif analysis was performed using HOMER; the top 10 ranked motifs are shown with their p-values. (J) RNA-seq-based expression of SOX family proteins in HT-29 cells (GSE108621). (K) Density plot of TRIM28 and SOX9 ChIP-seq datasets centered on 9412 TRIM28-occupied regions in HT-29 cells treated with DMSO or TSZ for 4 h. Each row represents a single peak. (L) Metagene representation of the ChIP-seq signal for SOX9 across 9412 TRIM28-occupied regions in HT-29 cells treated with TSZ for 4 h. Metagenes centered on the middle of 9412 regions and 10 Kb around the center of the TRIM28-occupied regions are displayed