Figure 3
From: Dysregulated fibronectin trafficking by Hsp90 inhibition restricts prostate cancer cell invasion
AUY922 induces expression of fibronectin, resulting in its cytosolic accumulation in punctate compartments. (A) Western blot analysis of the effect of 17-AAG or AUY922 on Hsp70, fibronectin (FN1) and cytoskeletal proteins, in LNCaP and PC-3 cells. Cells were treated with DMSO control or the indicated doses of AUY922, or 17-AAG and collected at 48 h. Full-length blots are presented in Supplementary Figure 6. To examine intracellular localization of FN1, we used (B) cellular fractionation of LNCaP cells treated as indicated for 48 h (W = whole cell lysate, C = cytoplasmic fraction, N = nuclear fraction; full length blots are presented in Supplementary Figure 6), and (C) fluorescence microscopy of LNCaP cells treated for with AUY922 (25 nM) for 48 h and probed for FN1 (antibody obtained from Professor Jane Sottile, Rochester University) followed by secondary ALEXA 488 with images taken using Leica SP8 confocal microscope at 40x magnification. (D) The number and distribution of FN1 particles in cultured LNCaP cells with AUY922 (25 nM) for 48 h was quantified by automated image analysis (CellProfiler) of fluorescence microscopy images captured with an INCell 2200 automated imaging system (GE Healthcare) at 20x magnification.
