Fig 2: A schematic diagram of the assembly of the CtIP-CtBP1/2-HDAC1-AP1 complex and its role in osteosarcoma tumorigenesis Overexpressed CtIP couples with CtBP1/2 heterodimer, HDAC1, and AP1 subunits to assemble a transcriptional complex. This complex binds to the promoters of MLH1, MSH3, BRCA1, and CDKN1A to repress their expression. The downregulation of these genes impairs the DNA repair process and increases genome stability, causing tumorigenesis.

Fig 3: Identification of CtIP-CtBP1/2-HDAC1-AP1 downstream targets by microarray analysis and conserved AP1 consensus sequence(A) Microarray results. RNA samples from Control-KD, CtIP-KD1, and CtBP1-KD1 cells (in Saos2 background), as well as Control-OE, CtIP-OE, and CtBP1-OE cells (in hFOB1.19 background) were subjected to microarray analysis. Genes that were consistently dysregulated in CtIP-KD1 and CtBP1-KD1 but conversely expressed in CtIP-OE and CtBP1-OE cells are presented. Data are presented as the mean ± SD of three independent experiments. (B) Prediction of AP1 binding sites on the promoters of potential target genes of CtIP-CtBP1/2-HDAC1-AP1. The promoters (2000 bp length in the upstream of ATG) of the genes shown in the figure were used to identify the AP1 binding sites (TGAG/CTCA).

Fig 4: CtBP loss activates caspase 8 via death receptor 4.a, b KURAMOCHI cells were infected with indicated shRNAs. Cell death receptor expression was examined by western blotting (a), DR4 and DR5 mRNA levels were examined by qPCR (b), **p < 0.01 as compared with shCtrl group. Arrow in FAS blot points to unmodified FAS, and higher molecular weight forms are consistent with known glycosylation58. c–e KURAMOCHI cells were treated with indicated siRNAs along with shRNAs. Cleaved caspase 8, and the knockdown efficiency of DR4 and CtBP1/2 was determined by immunoblotting (c); caspase 8 activity was examined by enzymatic assay (d), **p < 0.01 as compared with shCtrl group; and viable cells were counted by Trypan blue assay at indicated time points (e). ***p < 0.001 as compared with shCtrl group.

Fig 5: CtBP1 and CtBP2 coordinate to repress DR4/5.a Western blotting was performed to determine abundance of the indicated proteins in OVCA429 cells after infection with the indicated lentiviral shRNAs. b Fold induction of DR4/5 mRNAs in OVCA429 cells infected with the indicated lentiviral shRNAs was assessed by qPCR. c Western blotting to determine CtBP1/2 abundance in input and IP’s from ChIP experiments with OVCA429 cells when infected with indicated lentiviral shRNAs. d CtBP1/2 occupancy at DR4/5 promoters in OVCA429 cells. ChIP was performed with OVCA429 chromatin from cells in which CtBP1 or CtBP2 were knocked down with the indicated lentiviral shRNA’s. Indicated PCR amplicons were used for ChIP. e ChIP-reChIP assay showing CtBP1/2 occupancy at the indicated regions of the DR4/5 promotors. ChIP-reChIP was performed with OVCA429 chromatin and the first antibodies on the left hand side of the graphs, and second antibodies on the right hand side of each graph, **p < 0.01 as compared with normal IgG group.