Figure 3A. Schematic representation of the mechanism of action of dBRD9-A. dBRD9-A simultaneously recruits the ubiquitin E3 ligase Cereblon (CRBN) to the bromodomain (BD) of BRD9, thereby facilitating the ubiquitination and degradation of BRD9. B. A549 cells were treated for the indicated time with either DMSO or 125nM dBRD9-A. Following lysis and western blot analysis for the indicated proteins, BRD9 levels were quantified by densitometry and made relative to levels in DMSO-treated cells (% below). Data are representative of at least two biological replicates. C. A549 cells were treated with 125nM dBRD9-A (+) or DMSO (-) for 6h prior to lysis and analysis by western blot for the indicated proteins. Data are representative of at least two biological replicates. D. A549 cells were treated with 125nM dBRD9-A (+) or DMSO (-) for 6h prior stimulation with 1000 IU/mL of IFN-α2 for 16h. Following lysis, western blot was used to detect levels of the indicated proteins. Data are representative of at least two biological replicates. E. A549 cells were treated as described in (D), but following stimulation with 1000 IU/mL of IFN-α2 for 16h were infected with IAV (WSN/33) at an MOI of 0.01 PFU/cell. Viral titers were determined after 24h by plaque assay. Data represent means and standard deviations from n=3 biological experiments (individual data points shown). Statistical significance was determined by 1-way ANOVA on log-transformed plaque counts (***p-value < 0.001). Data information: dotted lines are a visual guide for maximum and minimum virus replication in control cells in the absence and presence of IFN-α2, respectively. Numbers above IFN-α2-treated bars indicate their approximate difference to the respective untreated conditions. F-H. A549 cells were treated as described in (E), but IFN-α2 concentrations ranged from 10-1000 IU/mL depending upon the virus, and infections were similarly performed but with: VSV-GFP (F); HSV1-GFP (G); or VSV-G pseudotyped HIV1-GFP (H). Data information: Total Integrated Green Fluorescent Intensities were determined using the Incucyte live-cell analysis system after 10h (G), 24h or 48h (H). Data represent means and standard deviations from n=3 biological experiments (individual data points shown). Statistical significance was determined by 1-way ANOVA on log-transformed intensity values (*p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001). dotted lines are a visual guide for maximum and minimum virus replication in control cells in the absence and presence of IFN-α2, respectively. Numbers above IFN-α2-treated bars indicate their approximate difference to the respective untreated conditions. I-L. Hep2 (I), Huh-7 (J), MRC-5 (K), and undifferentiated HTBE (L) were treated and infected with VSV-GFP Data information: Total Integrated Green Fluorescent Intensities were determined using the Incucyte live-cell analysis system after 24h Data represent means and standard deviations from n=3 biological experiments (individual data points shown). Statistical significance was determined by 1-way ANOVA on log-transformed intensity values (*p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001). , dotted lines are a visual guide for maximum and minimum virus replication in control cells in the absence and presence of IFN-α2, respectively. Numbers above IFN-α2-treated bars indicate their approximate difference to the respective untreated conditions.