Figure 4

SF-1 controls centrosome homeostasis independent of its DNA binding domain. (A) Fractionation of centrosome from FLAG-tagged SF-1 transfected Y1 cells by sucrose gradient ultracentrifugation. Immunoblots of proteins in fractionated cell extracts are shown. Cyt: cytoplasmic fraction; Nu: nuclear fraction; Sup: post-centrosomal supernatant; WCE: whole cell extract; γ-tub: γ-tubulin; hnRNAP: hnRNP A1 (nuclear marker) and Mito II: mitochondria complex II (cytoplasmic marker). (B) SF-1 overexpression inhibits the activation of DNA-PK in the centrosome. Centrosomal extracts of EYFP (control) or shRNA-resistant 3-FLAG-SF-1 (3 F-SF1) overexpressing shluc (luc) or shsf1#3 (#3) infected Y1 cells were analyzed by immunoblotting with antibodies against phosphorylated DNA-PKcs (pPKcs), phosphorylated Akt (pAkt), Akt, SF-1, and Ku70. For the immunoblot of SF-1, the upper band is exogenous 3 F-SF1; the lower band is endogenous SF-1 (C) Transcriptional activities of EYFP (negative control), wild-type SF-1 (positive control), or D70-SF-1 (D70) were measured in Y1 cells using human CYP11A1 2.3 k promoter linking to luciferase as a reporter. (D) Quantification of cells with multiple centrosomes in shluc (luc) or shsf1#3 (#3) infected EYFP, SF-1 or D70 expressing Y1 cells. These results are mean ± S.D. from three independent experiments; more than three hundred cells were measured in each individual group.