<i>Arid4b</i> germline amino acid sequence variants.

<p>Substitutions in the AKR allele are shown relative to a consensus sequence that is identical between DBA, FVB, and C57Bl/6. The+symbol indicates conserved substitutions.</p

Cellular processes regulated by <i>Arid4b</i> knockdown.

<p>Biological processes are listed in order of statistical significance after applying a cutoff based on z-scores with an absolute value greater than 2. Negative z-scores indicate downregulation; positive z-scores indicate upregulation.</p

High expression of <i>ARID4B</i> is associated with poor clinical outcome.

<p>In patients with ER-positive tumors who were node negative at diagnosis (A) distant metastasis-free survival (DMFS) was significantly lower (p = .009) in patients with high expression (blue) compared to middle (red) or low (gray) expression of <i>ARID4B</i>, and multivariate analysis of 440 cases (B) was performed to determine metastatic progression hazard ratios of 0.54 and 0.42 for median and low <i>ARID4B</i> terciles, respectively, compared to the high <i>ARID4B</i> tercile. The association of high <i>ARID4B</i> with poor DMFS was also highly significant (p = 3.05×10∧−4) among ER-positive patients in the absence of adjuvant therapy (C) with similar hazard ratios (D) of 0.53 and 0.49 for middle and low <i>ARID4B</i> groups compared to high <i>ARID4B</i>.</p

<i>Arid4b</i> expression is correlated with tumor growth and metastasis.

<p>Gene expression microarray data from 18 of the AKXD recombinant inbred strains shows expression of <i>Arid4b</i> is positively correlated with tumor burden (A) and metastatic density (B). The formula used to calculate metastatic density was ln[(metastases/um²)×10⁸].</p

ARID4B binds BRMS1.

<p>HA-BRMS1 was coprecipitated with FLAG-ARID4B (A) and FLAG-ARID4B was coprecipitated with myc-BRMS1 (B). No difference in binding to BRMS1 was observed between the AKR and DBA alleles of ARID4B.</p

Stable shRNA–mediated knockdown of <i>Arid4b</i> inhibits lung metastasis.

<p>ARID4B protein levels were analyzed by western blot to determine level of knockdown in cell lines stably expressing different <i>Arid4b</i> shRNAs. Experiment was performed in triplicate and a representative blot is shown in panel A. Lysate from 293 cells transiently transfected with <i>Arid4b</i> was used as a positive control (+C). Untransduced 6DT1 and scrambled control lines are designated “untr” and “scr” respectively. The lower non-specific (n.s.) band most likely represents ARID4A, based on a nearly identical epitope sequence and consistent with the observed difference in molecular weight. Expression of the non-specific band remained relatively unchanged across the panel of stable lines. Densitometry was performed to quantitate knockdown at the protein level in the <i>Arid4b</i> shRNA lines relative to the scrambled control line (B) Data are shown as the mean ± standard error of three independent experiments. Kruskal-Wallis followed by Conover-Inman post hoc tests were used to determine statistical significance versus the scrambled control cell line. *, p<.05; **, p<.01; ***, p<.001. Primary tumors were weighed (C) and lung surface metastases were counted (D) at 3.5 weeks after orthotopic implantation of 10∧5 cells from the scrambled control, H3, and H4 lines into the mammary fat pad of FVB mice (n = 10 per cohort). Horizontal bars represent median values and statistical significance was determined using Kruskal-Wallis tests followed by Conover-Inman correction for multiple comparisons.</p

Differential binding of the AKR and DBA variants of ARID4B to the mSIN3A complex.

<p>Western blots (A) show equivalent input of V5-ARID4B, mSIN3A, mSDS3, and equivalent pull-down of the two variants using an anti-V5 antibody. Decreased binding of the DBA variant of ARID4B to mSIN3A and mSDS3 was observed relative to the AKR variant. Experiment was performed in triplicate and densitometry analysis (B) revealed a 51% reduction in mSIN3A binding (p = .037) and a 37% reduction in mSDS3 binding (p = .026) for the DBA variant relative to AKR. Statistical significance was determined using paired t-tests. *, p<.05.</p