Immunoblots were probed using (A)+(B) anti GST and (C) anti Hsp90 antibodies. 160 and 320 nM) to immobilized Hsp90 (10 g.ml?1). Hsp90 was immobilized onto the surface of a ProteOn? GLM Sensor chip (#176-5012, Bio-Rad, US) and protein conversation was decided at 37C. Sensorgrams are representative of quadruple experiments at varying FN concentrations (E) Saturation binding curve of FN-Hsp90 SPR binding data. Req values were plotted against concentration and fit with a non-linear curve (one site binding). An R2 value equal to 0.9800 was reported.(TIF) pone.0086842.s001.tif (1.0M) GUID:?C48694B2-000E-46EA-89EE-4513039B68AF Physique S2: Validation of the deoxycholate (DOC) assay to quantitate FN matrix assembly in Hs578T breast malignancy cells. Confluent Hs578T cells were incubated with increasing concentrations of the MEK1/2 Pllp inhibitor (U1026) for Chrysin 16 hours at 37C. Cells were lysed and DOC-soluble and DOC-insoluble FN fractions isolated using the explained DOC assay and relative levels determined by immunoblotting. Analysis of GAPDH levels exhibited no contamination between the soluble and insoluble fractions.(TIF) pone.0086842.s002.tif (305K) GUID:?8D0E74DB-E034-4135-8A92-D25106461B89 Figure S3: Etoposide has no effect on the extracellular FN matrix. Adherent Hs578T cells were treated with etoposide (0.5 M or 1 M) and the effect around the FN matrix examined by Chrysin confocal microscopy. The IC50 value (0.96 M) for etoposide has previously been determined in the Hs578T cell collection . Duplicate images of etoposide (0.5 M) treatment are shown. Level bars are equivalent to 50 m.(TIF) pone.0086842.s003.tif (1.9M) GUID:?738807B7-2291-402F-8E31-7F7B321C6E7E Physique S4: Analysis of fibronectin dynamics using exogenous fluorescent FN. (A) Cell imaging of exogenously added fluorescently labeled FN (FN-550). Hs578T cells were produced in phenol-red free media in sterile glass-bottomed microscopy culture dishes and allowed to undergo fibrillogenesis in media supplemented with FN-550 (50 nM). Fixed cells were stained using mouse anti human FN followed by donkey anti mouse DyLight? 488 fluorescent secondary antibodies. Images were captured using the Zeiss LSM 510 Meta confocal microscope. Level bars are equivalent to 20 m. White arrows show regions of incorporation of the exogenous FN-550 into the extracellular FN matrix. (B) Confluent Hs578T cells were allowed to undergo fibrillogenesis in media supplemented with FN-550 (50 nM). Following the confirmation of a fluorescent extracellular FN matrix, cells either remained untreated or were treated with novobiocin (NOV; 1 mM) for a period of 2, 4 or 8 hours. Cells were fixed and images captured using confocal microscopy. Three images were captured for each treatment in areas where comparable cell numbers were observed. Scale bars are equivalent to 20 m. Data are representative of three impartial experiments with comparable results.(TIF) pone.0086842.s004.tif (3.9M) GUID:?96B7FCC0-CE6F-479C-911F-DA6B1E8251A7 Abstract Chrysin Heat shock protein 90 (Hsp90) has been recognized in the extracellular space and has been shown to chaperone a finite quantity of extracellular proteins involved in Chrysin cell migration and invasion. We used chemical cross-linking and immunoprecipitation followed by tandem mass spectrometry (MS/MS) to Chrysin isolate a complex containing Hsp90 and the matrix protein fibronectin (FN) from breast cancer cells. Further analysis showed direct binding of Hsp90 to FN using an co-immunoprecipitation assay, a solid phase binding assay and surface plasmon resonance (SPR) spectroscopy. Confocal microscopy showed regions of co-localisation of Hsp90 and FN in breast malignancy cell lines. Exogenous Hsp90 was shown to increase the formation of extracellular FN matrix in the Hs578T cell line, whilst knockdown or inhibition of Hsp90 led to a reduction in the levels of both soluble and insoluble FN and could be partially rescued by addition of exogenous Hsp90. Treatment of cells with novobiocin led to internalization of FN into vesicles that were positive for the presence of the lysosomal marker, LAMP-1. Taken together, the direct interaction between FN and Hsp90, as well as the decreased levels of both soluble and insoluble FN upon Hsp90 inhibition or knockdown, suggested that FN may be a new client protein for Hsp90 and that Hsp90 was involved in FN matrix assembly and/or stability. The identification of FN as a putative client protein of Hsp90 suggests a role for Hsp90 in FN matrix stability, which is important for a number of fundamental cellular processes including embryogenesis, wound healing, cell migration and metastasis. Introduction Heat shock protein 90 (Hsp90) is one of the most abundant and ubiquitously expressed chaperone proteins, constituting approximately 1C2% of the total cell protein complement , , , . Playing a vital role in cell survival, Hsp90 modulates the stability and transport of a.