IRF1 mRNA becomes underrepresented in MEN1 knockdown cells. (a) Northern blot analysis of RNA collected from shRNA-MEN1 (shMEN1) and shRNA-NS (NS) cells, induced with IFN-γ for 2 hours. The blot was probed with 32P-labeled IRF1 and GAPDH (loading control) cDNA probes. Quantification was done with the Storm 840 Imager and ImageQuant TL. (b) RT-PCR of RNA collected from shRNA-MEN1 and shRNA-NS cells, using primers designed to exon 4 (forward) and exon 5 (reverse), to specifically amplify IRF1 mRNA. β-actin primers were used as a loading control. (c) Western blot of extracts prepared from shRNA-MEN1 and shRNA-NS cells uninduced or induced with IFN-γ for indicated times. An IRF1 antibody was used to develop the blots. GAPDH served as a loading control. (d) Graphical representation of western blots (n = 3) as in panel C. IRF1 protein levels in the two cell lines were first normalized to GAPDH and then shRNA-MEN1 IRF1 levels were compared to shRNA-NS IRF1 levels. Quantification was performed using Image J. Error bars are standard error. (e) Cytopathic effect of VSV in the shRNAmir-MEN1 cell line. Cells were cultured with (+) or without (-) doxycycline, treated with IFN-γ, IFN-α or left untreated for 12 hours and infected with dilutions of VSV. One of two biological replicates is shown. (f) qRT-PCR to detect the ratio of unspliced to total IRF1 RNA at the indicated times after IFN-γ induction (n = 4). The shRNA-MEN1 ratio is presented in reference to the shRNA-NS ratio, which was set to 1 at each condition. Error bars are standard error. **P ≤ 0.01, *P ≤ 0.05. IFN: interferon; MEN1: multiple endocrine neoplasia type 1; NS: nonsilencing; RT-PCR: real time PCR; VSV: vesicular stomatitis virus.