Fig 2: Ajuba, DBC1 and CBP/p300 forms a ternary complex to enhance ERα target gene transcription. (A) Plasmids were transfected into 293T cells and the Co-IP assay was performed by using Flag-M2 beads (the relative amount of immunoprecipitated HA-CBP was semi-quantified by grayscale analysis and the mean values of the three repeats were labeled). (B) Myc-Ajuba, HA-CBP and Flag-DBC1 plasmids were co-transfected into 293T cells and sequential co-IP assays were performed. The first round co-IP assay was carried out by using Flag antibody or normal IgG and then the 3XFlag-peptides eluted product was subjected to the second round co-IP assay by using HA antibody or normal IgG. (C) The endogenous co-IP assay was performed in T47D-PCDH-Vector or T47D-PCDH-Myc-Ajuba cells by using ERα antibody. (D) siRNA-DBC1 was transfected into T47D cells for 48 h, and the co-IP assay was performed by using ERα antibody. (E) The siRNA-NC or siRNA-DBC1 was transfected into T47D cells and the ChIP assay was performed after the E2-treatment and the results were normalized to input and 5–10% as input (three repeats, * means P < 0.05). (F) TFF1-luciferase reporter assays were performed in 293T cells. Transfected cells were treated with 20 nM E2 for 12 h before harvesting and the value was normalized to β-gal (three repeats, * means P < 0.05). (G) The model of Ajuba regulating ERα transcriptional activity. Ajuba forms a complex with DBC1 and CBP to enhance the acetylation and transactivity of ERα.

Fig 3: KLLN’s role in p53 acetylation after DNA damage is through the regulation of DBC1 phosphorylation.a Immunoblotting for Thr454-phosphorylation after doxorubicin-induced DNA damage showed that knocking down KLLN expression resulted in decreased DBC1 phosphorylation in MCF10A (i) and MCF7 (ii) cells. b Immunoblotting for Lys382-acetylation of p53 after doxorubicin-induced DNA damage showed decreased p53 acetylation after knockdown of KLLN expression in MCF7 (i) and MCF10A (ii) cells

Fig 4: DBC1 Depletion Decreases p53 Half-Life in U2OS Cells(A) U2OS cells treated with 100 μM cycloheximide (Sigma) for up to 90 min were harvested for immunoblotting using anti-p53 antibody. See also Figure S3.(B) Quantification of relative p53 protein level post-cycloheximide treatment. (Left) Error bars represent mean ± SD from three independent experiments. (Right) Derived p53 half-life is displayed in the table.(C) Immunoblotting of lysates from (A).(D) p53 IPs from cytoplasmic and nuclear fractions of U2OS cells treated with control, CBP, DBC1, or CBP-DBC1 siRNA, followed by immunoblotting with anti-p53 and ubiquitin antibodies.(E) Immunoblotting of cytoplasmic and nuclear lysates from (D).

Fig 5: DBC1 Overexpression Promotes p53-Dependent Apoptosis(A–C) Representative flow cytometry histograms for apoptosis using U2OS cells (A), MCF-7 cells (B), and MCF-10A cells (C). Analysis of apoptosis by flow cytometry of annexin-PI-stained U2OS, MCF-7, and MCF-10A cells transfected with either empty vector or myc-DBC1 and treated with 25 μM cisplatin. Percentages in the Q2 and Q4 quadrants represent the early and late apoptotic populations, resepctively. See also Figure S6B.(D–F) Quantification of apoptosis measured by flow cytometry from three independent experiments for U2OS cells (D), MCF-7 cells (E), and MCF-10A cells (F) treated as in (A)–(C). Error bars represent mean ± SEM from three independent experiments.(G–H) U2OS cells (G), MCF-7 cells (H), and MCF-10A cells (H) transfected with either empty vector or myc-DBC1 were treated with 25 μM cisplatin, followed by cell lysis and immunoblotting using the indicated antibodies. (I) U2OS cells were treated with either control siRNA or p53 siRNA, followed by transfection with empty vector or full-length myc-DBC1 plasmids. 24 h before lysis, cells were exposed to 25 μM cisplatin. Lysed cells were subjected to immunoblotting using the indicated antibodies.