Figure 8. IFITM1 knockdown increases p21 expression through enhanced STAT1 activity, which mediates cell death.

(a) Cell lysates from MCF-7:5C cells transfected with scrambles control siRNA (siCon) or three different IFITM1 siRNA sequences (siIFITM1 A, B, C) were immunoblotted for IFITM1, p53 and p21 expression. (b) Dual Annexin V/Propidium Iodide staining was used to quantify cell death in each transfection group as compared to siCon. Data represent means ± SD from two experiments conducted in duplicate. (c) Activity at the p21 promoter was determined by luciferase assay. The motifs of interest are indicated in the promoter map (upper panel). MCF-7:5C cells were transfected with p21 luciferase reporter and renilla reporter constructs with siCon or siIFITM1. Cells were treated with 10μM Rux or vehicle (Con) and assayed after 24 hours of Rux treatment. Values represent means ± SD of two independent experiments done in triplicate. (d) MCF-7:5C cells were transfected with STAT/GAS luciferase cocktail and with siCon or siIFITM1 or treated with 10μM ruxolitinib (Rux). (e) Binding of STAT proteins to p21 promoter. Fixed cell lysates from MCF-7:5C/shIF cells treated with vehicle (−Dox) or 1μg/mL doxycycline (+Dox) for 24 hours and subjected to chromatin immunoprecipitation (ChIP) with antibodies against STAT1, STAT2, STAT3 or rabbit IgG control. qPCR was performed on the isolated DNA using primers designed to amplify the −640 STAT regulatory region in the p21 promoter. Recruitment was compared to input DNA and displayed as mean ± SD of technical triplicates. (f) MCF-7:5C cells were transfected with siCon, siIFITM1C, p21 siRNA (siP21) or siIFITM1-C with sip21 and lysates were immunoblotted for phospho-STAT2 (p-STAT2), STAT2, phospho-STAT1 (p-STAT1), STAT1, phospho-STAT3 (p-STAT3), STAT3 IFITM1, phopsoh-p21 (p-p21) and p21. * p<0.05, ** p<0.01