Fig 2: TP treatment inhibited NF-?B–mediated prosurvival signals induced by TNF-a. (A) Time-dependent NF-?B activation induced by TNF-a (5 ng/ml). (B) Relative NF-?B activity induced by TP (25 nM) and TNF-a (5 ng/ml), 30 min after TNF-a treatment. (C–D) Representative western blots and relative intensity of protein bands of NF-?B p65 nuclear protein, with Lamin B1 as the loading control, 30 min after TNF-a (5 ng/ml) treatment. (E–H) Representative western blots and relative intensity of protein bands of CIAP1, CIAP2, XIAP, and FLIP in AGS and MKN45 cells treated with TP (25 nM) and TNF-a (5 ng/ml), 24 h after TNF-a application, with ß-actin as the loading control. Results were expressed as mean ± SEM, and statistical analysis was performed using one-way ANOVA or two-way ANOVA followed by Tukey’s multiple comparison test. *, # p < 0.05 (n = 3). *p compared with the control group and # p compared with the TNF-a-treated group.

Fig 3: BRD7 negatively regulates the expression of BIRC2 by inhibiting the activation of the BIRC2 enhancer.A ChIP-PCR assays using antibody specific for Flag was performed to validate the BRD7-binding sites in the 6th intron of BIRC2, while a 400-bp fragment of the BIRC2 potential promoter region without BRD7 binding site was used as a negative control. B Western blotting using antibodies against BRD7 and BIRC2 was performed to confirm BRD7 and BIRC2 protein levels. GAPDH served as an internal control. C qRT-PCR assays were used to confirm BRD7 and BIRC2 mRNA levels. GAPDH served as an internal control. D The dual-luciferase reporter assays was performed to determine the effect of BRD7 on the promoter activity of BIRC2. E ChIP-PCR assay using antibodies specific for H3K4me1 was used to validate the H3K4me1 enrichment at the B7BS, and B7BS was divided into four PCR fragments of about 200 bp, each fragments was amplified with each pair of primers. (F) and (G) The dual-luciferase reporter assays were used to determine the enhancer activities of B7BS and mut-B7BS and the effect of BRD7 on their activities. The error bars are presented as the mean ± SD. ***P < 0.001; NS, no significance. All experiments were performed in triplicate.

Fig 4: BRD7 inhibits tumor metastasis in vivo through regulation of BIRC2 expression.A Representative image of macroscopic mouse lung tissue in the metastatic tumor model. B The number of metastatic lung nodules of every mouse per group was counted in microscopy. C H&E staining is shown in control, BRD7 overexpression, BIRC2 overexpression and BIRC2 restoration group. Red arrows indicate metastatic tumors, scale bar, 100 µm. Error bars represent the mean ± SD. **P < 0.01, ***P < 0.001.

Fig 5: BRD7 inhibits tumor growth in vivo through regulation of BIRC2 expression.A Growth curve of tumor xenografts. B Representative tumor images of the 5-8 F xenograft model in nude mice. C Tumor weight quantification. D IHC (DAB staining) for Ki-67, CDK4 and cleaved-PARP in the 5-8 F xenograft model. Three tumors were analyzed per group. Original magnification, 200×; scale bars represent 50 µm. Error bars represent the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.