Elevated level of intracellular IFNα drives constitutive overexpression of IFITM1. (A) ELISA analysis of baseline expression of IFNα in cell lysates and supernatant in MCF-7:5C and MCF-7 cells. Cells (1 x 106) were seeded in a six-well plate in their standard culture media and after 48 hours cells were harvested and the supernatant and pellets were collected. Cell pellets were then lysed by sonication in RIPA buffer containing protease inhibitors. IFNα was measured in the supernatants and cell lysates by ELISA as described in Methods. All the illustrated data were performed in duplicate and are expressed as mean values of three independent experiments ± SD. (B) Measurement of IFNα mRNA was determined by real-time PCR. Fold change was calculated by means of the ΔΔCT method using PUM1 as an internal control. Values are displayed as relative to MCF-7 cells and are means of triplicate measurements ± SD in three independent experiments. (C) Blockade of type 1 interferon receptor, IFNAR1, using neutralizing antibody MAB1155, in MCF-7 and MCF-7:5C cells. Cells were pretreated with 5 μg/mL anti-IFNAR1/2 for 4 hours and then treated with 20 U/mL human recombinant IFNα for 24 hours. Cells were analyzed by Western blot to assess IFITM1, PLSCR1 and β-actin protein level. Results shown are representative of three independent experiments. The protein levels were quantified using the ImageJ software (downloaded from NIH website ) and normalized as the ratio related to β-actin. *P <0.05 or **P <0.01 versus control. (D) siRNA knockdown of IRF-7 expression in MCF-7:5C cells. Cells were transiently transfected with siRNA targeting IRF-7 and after 24 hours knockdown was verified at the mRNA and protein level via RT-PCR and Western blot analyses. (E) Effect of IRF-7 knockdown on IFNα expression in resistant MCF-7:5C cells. Cells were transiently transfected with siRNA targeting IRF-7. After 24 hours, IFNα mRNA and IFNα protein expression were determined by real-time PCR and ELISA, respectively. Data shown for RT-PCR is expressed as fold change over cells transfected with control siRNA. Values are displayed as means ± SD of three independent experiments performed in triplicate. *P <0.05 or **P <0.01. (F) The effect of IFNα or IRF-7 knockdown on IFITM1, p-STAT2, STAT2, p-STAT1, STAT1, and IRF-7 protein expression in resistant MCF-7:5C cells. Cells were transiently transfected with siIFNα, siIRF-7 or siCon for 48 hours and Western blot analysis was performed on lysates. Results shown are representative of two independent experiments IFITM1, interferon induced transmembrane protein1; IFNAR1, IFN alpha Type 1 receptor; NIH, National Institutes of Health; PLSCR1, phospholipid scramblase 1; SD, standard deviation.