4.1. Cells and Reagents
MCF-7, MDA-MB-468 and MDA-MB-231 breast adenocarcinoma cells were maintained in DMEM with 1 g/L glucose, l-glutamine and sodium pyruvate formulation, supplemented with 10% FBS and 1% penicillin/streptomycin. HCC-1806, Hs578T and BT549 breast cancer cells were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. MCF-10A breast epithelial cells were maintained in MEGM containing 13 mg/mL BPE, 0.5 mg hydrocortisone, 10 μg/mL hEGF, 5 mg/mL insulin and 100 ng/mL Cholera toxin (Lonza, Allendale, NJ, USA). Normal HMEC (Primary Mammary Epithelial cells) cells were grown in MECM medium, supplemented with MEC growth kit (ATCC, Manassas, VA, USA). All cell lines were purchased from ATCC. They were cultured in humidified incubators at 37 °C in 5% CO2.
For plasmid transfection studies, 3xHA-ARRDC3, GFP-ARRDC3 and 3xHA-NEDD4 were purchased from the GeneCopoeia, Inc. (Rockville, MD, USA). pCMV-Myc was purchased from Agilent (Santa Clara, CA, USA). Integrin β4 cDNA was cloned into pEGFP-N2 and pCMV-Myc respectively. pHLuorin-CD63 was a gift from Dr. Maarten Bebelman (Vrije University, Amsterdam, Netherlands). The transfection of all plasmids was carried out using Lipofectamin LTX-Plus (Invitrogen, Grand Island, NY, USA) or Lipofectamin 3000 (Invitrogen).
Integrin β4 (sc-9090), HSP70 (sc-24), Dicer (sc-30226) and β-actin (sc-1615) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). GFP monoclonal antibody was obtained from Clontech (Mountain View, CA, USA). ARRDC3 (ab64817), Rab5 (ab109534), TSG-101 (ab30871), CD63 (ab68418) and CD9 (ab3223) antibodies were obtained from Abcam (Cambridge, MA, USA). CD104 (BD555719), Flotilin-1 (BD610820) and GM130 (BD610822) were purchased from BD Biosciences (San Jose, CA, USA). NEDD4 (#2740), Calnexin (#2679), Myc-Tag (#2272), HA-Tag (#2367 or #3724) antibodies were purchased from Cell Signaling.
MG 132 (proteasome inhibitors) and Cyclohexamide (CHX; protein synthesis inhibitor) were purchased from Sigma (St. Louis, MO, USA). EGF was obtained from Sigma-Aldrich and Lysotracker red DND-99 was purchased from Invitrogen.
siRNAs were purchased from Ambion (Thermo Fisher Scientific, Waltham, MA, USA) and used to target human integrin β4 (sense GCGACUACACUAUUGGAUUtt and antisense AAUCCAAUAGUGUAGUCG Ctg) and human NEDD4 (assay ID; s9417) Cells were plated on dish for 24 h before siRNA transfection. Transfection was performed using RNAiMAX reagent (Invitrogen) according to the manufacturer’s instructions. To enhance knockdown, cells were typically grown for 3 days. To generate stable NEDD4 knockdown cell lines, MDA-MB-231 cells were infected with lentiviruses expressing shRNA targeted against NEDD4 or GFP as control (Sigma). The infected cells were then selected by puromycin (20 μg/mL).
4.2. Western Blot Analysis
Cells were lysed in cold radioimmunoprecipitation assay–ethylenediaminetetraacetic acid (RIPA–EDTA) buffer (50 mM Tris, pH 7.4; 150 mM NaCl; 1% NP-40; 0.5% sodium deoxycholate; 0.1% SDS; and 5 mM EDTA) containing 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, and protease inhibitor (Thermo Scientific Pierce, Rockford, IL, USA). The protein concentrations were determined using the BCA protein assay kit (Thermo Scientific Pierce). The samples were separated on 4% to 20% gradient SDS PAGE and transferred to polyvinylidene difluoride (PVDF) membranes by using the Trans-Blot Turbo transfer system (Bio-Rad, Hercules, CA, USA). The blots were incubated with primary antibodies in TBS-T or TBS-T with 5% w/v nonfat dry milk, then with appropriate secondary antibodies conjugated to IgG-horseradish peroxidase. Proteins were detected using the Clarity Western ECL blotting substrate (Bio-Rad). All bands were imaged with ChemiDoc Touch Imaging System (Bio-Rad).
4.4. Assays That Monitor Intracellular Trafficking of ITG β4
4.4.1. Biotin-IP Based Integrin Recycling Assay
Cells were grown to 80–90% confluence in a 6-well plate were placed on ice and washed twice with ice cold PBS. Cell surface proteins were labeled with 0.5 mg/mL NHS-SS-biotin (Thermo Fisher Scientific) in PBS at 4 °C for 40 min. Unbound biotin was gently washed away in cold PBS containing 100 mM glycine. Labeled cells were transferred to pre-warmed serum-free medium and incubated at 37 °C for 30 min to allow internalization. After two washes with cold PBS, surface-remaining biotin was removed by cleavage with 100 mM MesNa (sodium 2-mercapto-ethanesulfonate) at 4 °C for 25 min. Cells were washed with ice-cold PBS and excess biotin was quenched with 5 mg/mL iodoacetamide for 10 min on ice. To allow integrin recycling, MesNa treated cells were incubated at 37 °C for different times (5, 20, 50 min). The cells were returned to ice, and biotin at the cell surface was removed by MesNa treatment. After washing with cold-PBS, cells were lysed in RIPA buffer and then biotinylated proteins were isolated from cells extract by immunoprecipitation on streptavitin-agarose beads (GE Healthcare, Chicago, IL, USA). Washed beads were eluted with SDS sample buffer and eluted proteins were subjected to immunoblotting analysis.
4.4.2. Immunofluorescence-Based Integrin Recycling Assay
Cells were grown on coverslips and serum starved at 37 °C overnight. The cells were transferred to ice to cool down. Surface integrin β4 were labeled with anti-Alexa 488-CD104 antibody (Invitrogen, #MA5-23641) in serum-free medium containing 0.01% bovine serum albumin (BSA) for 1h at 4 °C. Labeled cells were washed two times with cold serum-free medium containing 0.01% BSA and subsequently transferred to prewarmed free-serum medium. Integrin internalization was allowed at 37 °C for 2 h. For recycling, internalized integrin was stimulated with EGF for different times at 37 °C. The cells were washed twice with PBS, fixed in paraformaldehyde (PFA) and mounted with Fluoromount-G (Southern Biothech, Birmingham, AL, USA) or Vectashield DAPI (Vector lab, Burlingame, CA, USA) for. Integrin trafficking was monitored by immunofluorescence microscope. Protein localization was captured at 60× oil magnification and DIC was captured at CFI Plan Apo Lambda 60× Oil magnification using a Nikcon Eclipse Ts2R microscope with Nikon DSQi2 Digital Camera. All images were analyzed using NIS-Elements software (NIS-Elements advanced research 4.5 version, Nikon, Tokyo, Japan) and processed using Adobe Photoshop software (Adobe Photoshop CC 2015).
4.5. EVs Purification
For size exclusion chromatography (SEC) methods, cells were grown to 60–70% confluences, washed with PBS and incubated with a minimal volume of serum-free medium required to cover the cells. After 48 h, cell supernatant was centrifuged (350 g for 10 min and 2000 g for 30 min) to remove cells and debris, followed by filtering with 0.22 μm filter (Millipore, Burlington, MA, USA). The cell-free supernatant was concentrated to 100–200 μL with using Amicon Ultra-4 10 kDa filter (Millipore). For EV (mostly exosome) isolation, 100 μL of concentrated cell supernatants were subjected to SEC by qEV column (IZON Science Ltd., Cambridge, MA, USA). Briefly, the column was rinsed with 10 mL of filtered PBS before use. The samples were layered onto the top of qEV column followed by elution with PBS. The first 5 fractions (total 1 mL) were discarded (this void volume does not contain EVs). Subsequently, each 200 μL fraction (fractions from 6 to 17) was collected. The isolated EV samples were frozen at −80 °C. 7–9 fractions containing high EVs were pooled and filtered for subsequent assay. The size, concentration and Zeta-potential of EVs were measured using ZetaView (Particle Metrix, Germany) and NanoSight (Malvern Panalytical, UK) nanoparticle tracking analysis.