BRD9 is a Druggable Component of Interferon-Stimulated Gene Expression and Antiviral Activity (2021)
Figure 2

Figure 2. BRD9 is Important for Interferon-Stimulated Gene Transcription and Antiviral Activity.

A. An A549-derived BRD9-KO cell-clone (KO#1) was generated using a crRNA targeting exon 8 of BRD9. The target sequence of the crRNA (termed crRNA1), and the resulting genomic alterations determined by NGS for the two BRD9 alleles, are shown in comparison to an unedited control clone (CTRL#1). Generated indels lead to a premature termination codon (PMT) in the following exon. Encoded amino-acids are shown below the CTRL nucleotide sequence.

B. Western blot analysis of lysates from CTRL#1 or BRD9-KO#1 cells. BRD9 and β-actin were detected with specific antibodies. Data are representative of at least two biological replicates.

C. Western blot analysis of CTRL#1 or BRD9-KO#1 lysates from cells treated for the indicated times with 1000 IU/mL of IFN-α2. The indicated proteins were detected with specific antibodies. Data are representative of at least two biological replicates.

D. Immunofluorescence analysis of CTRL#1 or BRD9-KO#1 cells treated for the indicated times with 1000 IU/mL of IFN-α2. The indicated proteins were detected with specific antibodies. Data are representative of at least two biological replicates. Scale bars indicate 10μm.

E. RT-qPCR analysis of MX1 levels in CTRL#1 or BRD9-KO#1 cells following treatment, or not, with 100 IU/mL of IFN-α2 for 6h. GAPDH transcript levels were used for normalization. Data represent means and standard deviations of fold expression changes (relative to CTRL#1 without IFN-α2 treatment) from n=3 biological replicates (individual data points shown). Statistical significance was determined by unpaired 2-tailed t-test on ΔCt values (***p-value < 0.001).

F. Western blot analysis of CTRL#1 or BRD9-KO#1 lysates from cells treated or not for 16h with 1000 IU/mL of IFN-α2. The indicated proteins were detected with specific antibodies. Data are representative of at least two biological replicates.

G. CTRL#1 or BRD9-KO#1 cells were treated, or not, with 1000 IU/mL of IFN-α2 for 16h prior to infection with IAV (WSN/33) at an MOI of 0.01 PFU/cell. Viral titers were determined after 24h by plaque assay.

H. CTRL#1 or BRD9-KO#1 cells were stably-transduced with BRD9-expressing, or control (EV, empty vector), lentiviruses and treated with a range of IFN-α2 concentrations (0, 10, 100, 1000 IU/mL) for 16h prior to lysis and analysis for the indicated proteins by western blot. Data are representative of at least two biological replicates.

I. CTRL#1 or BRD9-KO#1 cells were stably-transduced with BRD9-expressing, or control (EV, empty vector), lentiviruses and treated with 1000 IU/mL of IFN-α2 for 16h prior to infection with IAV (WSN/33) at an MOI of 0.01 PFU/cell. Viral titers were determined after 24h by plaque assay.

Data information: For (G) and (I), 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; ****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. See also Figure EV2.