0-6 mole) dissolved in 3 mL methanol. This mixture was stirred under nitrogen while sealed with a polypropylene cap for 48 hours at room temperature. The resulting mixture was purified using a Sephadex LH-20 column (10 g of resin in methanol) and eluted with methanol. Fractions containing dendrimer were collected and evacuated using high-speed vacuum to give a constant weight of 40 mg dendrimer (DDN) or dendrimer-DBeQ (DDNDBeQ) formulation product. Transmission electron microscopy (TEM) was used to determine the dendrimer size and shape. Briefly, dendrimer were drop-coated on a carbon-coated copper grid for TEM-based size measurement and analysis as recently described [13].Transwell Invasion AssayH1299 cells were seeded and directly treated on a T-25 cell culture flask (Fisher Scientific). Following a 24-hour treatment of either empty dendrimer, dendrimer encapsulated DBeQ (50M) or vehicle-control PBS, the cells were trypsinized and counted. These treated cells were plated (10,000/well) onto matrigel (Corning, 200g matrigel per 400L of serum free media) coated transwell insert (0.4m pores, Corning). Cells were order Bay 41-4109 allowed to migrate for 24 hours under indicated treatments and transwell (blue) were counted using a Nikon Eclipse TS100 inverted light microscope and data is shown as mean ?SEM [1].ImmunoblottingH1299 cells were treated on six well plates with either NMS-873 (25M or 50M), DBeQ (25M or 50M) or left untreated. After 24 hours of treatment, whole cell Bayer 41-4109 supplement protein extracts were obtained by adding RIPA buffer, supplemented with 0.5 M EDTA and 1x HaltTM Protease inhibitor cocktail (Thermo Fisher) to each well. The proteins were separated using SDS-PAGE and then immunoblotted onto nitrocellulose membrane. The ubiquitin (Santa Cruz Biotechnology, 1:1000), NFB (Santa Cruz Biotechnology, 1:1000) and -actin (equal loading control, Sigma, 1:10,000) antibodies were used as primary antibodies, while goat anti-mouse IgG HRP and goat anti-rabbit IgG HRP were used as a secondary antibodies (1:6000, Amersham). The membranes were visualized using the ClarityTM Western ECL Blotting substrate (Bio-Rad) and C-DiGit Blot Scanner (LI-COR). The same protocol was used when comparing empty-dendrimer (DDN), DBeQ (50M), dendrimer encapsulated DBeQ (DDNDBeQ, 50M), and vehiclePBS control with the exception of a longer treatment period (48 hours) in order to better detect long term effects of proteins involved in tumor growth and progression.PLOS ONE | DOI:10.1371/journal.pone.0158507 July 19,4 /Dendrimer-Based Proteostasis-Inhibition in NSCLCImmunofluorescence Staining and MicroscopyH1299 cells were plated onto a 12-well plate and treated with either dendrimer (DDN), dendrimer encapsulated DBeQ (DDNDBeQ, 50M) or vehicle PBS control. After 24 hours of treatment, cells were fixed using 4 -paraformaldehyde and permeabilized with Triton-X 100, 0.5 ). The fixed cells were immunostained with ubiquitin primary antibody (1:500 dilution in 0.5 goat serum, Santa-Cruz Biotechnology). After 1 hour, secondary goat anti-mouse IgG-TR Texas Red antibody (1:1000 dilution in 0.5 goat serum) was added and incubated in the dark for another hour. Hoechst stain (0.5g/mL) was used to identify nuclei and images were captured using the ZOETM Fluorescent Cell Imager. The data from Ub-immunostaining and Ubiquitin-RFP transfected cells is shown as mean ?SEM of ubiquitin-positive cells. The same protocol was utilized for the co-staining of Ubiquitin and KDEL. The primary antibodies used were.0-6 mole) dissolved in 3 mL methanol. This mixture was stirred under nitrogen while sealed with a polypropylene cap for 48 hours at room temperature. The resulting mixture was purified using a Sephadex LH-20 column (10 g of resin in methanol) and eluted with methanol. Fractions containing dendrimer were collected and evacuated using high-speed vacuum to give a constant weight of 40 mg dendrimer (DDN) or dendrimer-DBeQ (DDNDBeQ) formulation product. Transmission electron microscopy (TEM) was used to determine the dendrimer size and shape. Briefly, dendrimer were drop-coated on a carbon-coated copper grid for TEM-based size measurement and analysis as recently described [13].Transwell Invasion AssayH1299 cells were seeded and directly treated on a T-25 cell culture flask (Fisher Scientific). Following a 24-hour treatment of either empty dendrimer, dendrimer encapsulated DBeQ (50M) or vehicle-control PBS, the cells were trypsinized and counted. These treated cells were plated (10,000/well) onto matrigel (Corning, 200g matrigel per 400L of serum free media) coated transwell insert (0.4m pores, Corning). Cells were allowed to migrate for 24 hours under indicated treatments and transwell (blue) were counted using a Nikon Eclipse TS100 inverted light microscope and data is shown as mean ?SEM [1].ImmunoblottingH1299 cells were treated on six well plates with either NMS-873 (25M or 50M), DBeQ (25M or 50M) or left untreated. After 24 hours of treatment, whole cell protein extracts were obtained by adding RIPA buffer, supplemented with 0.5 M EDTA and 1x HaltTM Protease inhibitor cocktail (Thermo Fisher) to each well. The proteins were separated using SDS-PAGE and then immunoblotted onto nitrocellulose membrane. The ubiquitin (Santa Cruz Biotechnology, 1:1000), NFB (Santa Cruz Biotechnology, 1:1000) and -actin (equal loading control, Sigma, 1:10,000) antibodies were used as primary antibodies, while goat anti-mouse IgG HRP and goat anti-rabbit IgG HRP were used as a secondary antibodies (1:6000, Amersham). The membranes were visualized using the ClarityTM Western ECL Blotting substrate (Bio-Rad) and C-DiGit Blot Scanner (LI-COR). The same protocol was used when comparing empty-dendrimer (DDN), DBeQ (50M), dendrimer encapsulated DBeQ (DDNDBeQ, 50M), and vehiclePBS control with the exception of a longer treatment period (48 hours) in order to better detect long term effects of proteins involved in tumor growth and progression.PLOS ONE | DOI:10.1371/journal.pone.0158507 July 19,4 /Dendrimer-Based Proteostasis-Inhibition in NSCLCImmunofluorescence Staining and MicroscopyH1299 cells were plated onto a 12-well plate and treated with either dendrimer (DDN), dendrimer encapsulated DBeQ (DDNDBeQ, 50M) or vehicle PBS control. After 24 hours of treatment, cells were fixed using 4 -paraformaldehyde and permeabilized with Triton-X 100, 0.5 ). The fixed cells were immunostained with ubiquitin primary antibody (1:500 dilution in 0.5 goat serum, Santa-Cruz Biotechnology). After 1 hour, secondary goat anti-mouse IgG-TR Texas Red antibody (1:1000 dilution in 0.5 goat serum) was added and incubated in the dark for another hour. Hoechst stain (0.5g/mL) was used to identify nuclei and images were captured using the ZOETM Fluorescent Cell Imager. The data from Ub-immunostaining and Ubiquitin-RFP transfected cells is shown as mean ?SEM of ubiquitin-positive cells. The same protocol was utilized for the co-staining of Ubiquitin and KDEL. The primary antibodies used were.