Mesna

Biotin-based assays were performed as described previously10 (link),13 (link). MDA-MB-231 or PC-3 cells were grown in 10% FBS-containing medium on 6 cm dishes to 80% confluence. The cells were placed on ice and washed once with cold PBS. Cell surface proteins were labelled with 0.5 mg/mL of EZ-link cleavable sulfo-NHS-SS-biotin (#21331; Thermo Scientific) in Hanks' balanced salt solution (H9269; Sigma) for 30 min at 4°C. Unbound biotin was washed away with cold medium, and prewarmed 10% serum-containing medium was added to cells. The biotin-labelled surface proteins were allowed to internalize at 37°C for the indicated time-points, after which the cells were placed quickly back on ice with cold medium. In the case of recycling, the cells were incubated at 37°C for 30 min to allow the internalization of all biotin-labelled integrins. The remaining biotin at the cell surface after internalization was removed with 60 mm MesNa (63705; sodium 2-mercaptoethanesulfonate: Fluka) in MesNa buffer (50 mm Tris–HCl, pH 8.6, 100 mm NaCl) for 30 min at 4°C, followed by quenching with 100 mm iodoacetamide (Sigma) for 15 min on ice. To detect the total amount of surface biotinylation, one of the cell dishes was left on ice after biotin labelling followed by treatment without reducing MesNa. Cells were washed with PBS and in the case of recycling, the cells were incubated again at 37°C for the indicated time-points followed by reduction and quenching of the recycled biotin-labelled surface molecules as above. The cells were lysed by scraping in lysis buffer [1.5% octylglucoside, 1% NP-40, 0.5% BSA, 1 mm EDTA with phosphatase and protease inhibitor coctails (Roche)] and incubation at 4°C for 20 min. Cell extracts were cleared by centrifugation (16 000 × g, 10 min, 4°C), and the biotinylated integrins were immunoprecipitated from the supernatant with 2 ng/μL of anti-β1 integrin antibody (CD29 K20; Custom Design Service, Beckman Coulter) and protein G sepharose beads (17-0618-01; GE Healthcare). Internalized integrins were detected from immunoblots with horseradish peroxidase (HRP)-conjugated anti-biotin antibody (#7075; Cell Signaling Technology), and after stripping the immunoblot, the total amount of immunoprecipitated integrins was detected with anti-β1 integrin antibody (MAB2252, Chemicon, or 610468, BD Transduction Laboratories). Enhanced chemiluminescence-detected biotin and β1 integrin signals were quantified as integrated densities of the protein bands with ImageJ (version 1.43u), and each biotin signal was normalized to the corresponding integrin signal. In the case of internalization, the relative amounts of biotin-labelled integrins inside the cell were normalized to the biotin signal of all surface labelled integrins, while in the case of recycling, the relative biotin signals were normalized to the corresponding biotin signal of all internalized integrins. The decreasing amount of biotin-labelled integrins inside the cell was inverted to represent the increase of β1 integrin recycling. The ELISA-based endocytosis assays were performed as described above. To determine the amount of internalized, biotinylated integrins, the cell lysate was incubated in a 96-well plate using the well-coating anti-β1 integrin antibody K20 and an HRP-coupled anti-biotin antibody for ELISA detection.

The construct pTYB-HAUb, comprising the sequences of the human Ub (lacking Gly 76), an intein and a chitin binding domain, plus an HA tag, was used to synthesize HAUb75-MESNa as described previously (Borodovsky et al., 2002 (link)). Briefly, Ub–intein–chitin domain fusion protein was expressed in Escherichia coli (18 h induction with 0.4 mM IPTG at 17°C). Cell pellets were resuspended in 50 mM HEPES, pH 7.4, 150 mM NaCl, and 0.5 mM TCEP and lysed in a high-pressure homogenizer. The cleared cell extract was loaded onto a 15 ml chitin bead (New England Biolabs) column at a flow rate of 0.5 ml/min. The column was washed with 60 ml of lysis buffer followed by 25 ml of lysis buffer containing 50 mM β-mercaptoethanesulfonic acid sodium salt (MESNa) and incubated overnight at 37°C for the induction of on-column cleavage. HAUb75-MESNa thioester was eluted with 25 ml of lysis buffer and concentrated: approximately 2.5 mg of protein was recovered from a 1-L culture. The N-terminal Met of the HA-tag was frequently processed off during expression, resulting in a mixture of two proteins that behaved identically in labeling experiments.
To synthesize the HA-UbC2Br or HA-UbPA probes, 0.2 mM of 2-bromoethylamine or 250 mM propargylamine was added to a solution of HAUb75-MESNa (1–2 mg/ml) in 500 μl of column buffer, respectively. pH was carefully adjusted to 8 with NaOH, and after 20 min shaking at 1,400 rpm, at room temperature, 100 μl of 2.0 M aqueous HCl was added and the resultant reaction mixture was promptly transferred to a PD10 gravity column for buffer exchange, according to the manufacturer's instructions.
The probe was then aliquoted and frozen at −80°C for storage (no significant deterioration is observed for several months of storage except for HA-UbC2Br, which is prone to hydrolysis). All HA-Ub-derived probes were analyzed by liquid chromatography mass spectrometry (LC-MS) using a 1290 UPLC (Agilent) coupled to a 6560 quadrupole time-of-flight (QToF) mass spectrometer (Agilent) to monitor the reaction and the product detected by [M+H]⁺ = 10,197.6221, with >90% purity.

Surface biotinylation: Cells were washed twice with cold PBS and incubated 0.3 mg/ml EZ-Link NHS-biotin (Thermo Scientific 21328) in PBS for 1 h on ice. Remaining biotin was quenched with 50 mM Tris-PBS pH 8. After lysis and sonication (see aggregation assay above) lysate was incubated with Neutravidin agarose resin (Thermo Scientific 29204) for 50 min at 4 °C. Beads were washed and eluted with Laemmli buffer.
Endocytosis assays: Cell were labelled as above and returned to medium + 1% BSA at 37 °C for the period of the endocytic assay. Cell were returned to ice and surface biotin was removed 50 mM MESNa (Sigma M1511) in PBS for 2× 10 min. Excess MESNa was quenched with cold 20 mM Iodoacetamide (in PBS) for 5 min. Cells were washed with PBS and lysed as described above. A variation on this was where aggregates were pelleted, and after solubilized+ sonication, biotinylated protein (that had been aggregated) were isolated with Neutravidin agarose resin.

Prior to ASCT on day 0, patients received high-dose chemotherapy with a combination of thiotepa (250 mg/m² per day, from day 9 to day 7), busulfan (3.2 mg/kg per day, from day 6 to day 5, and 1.6 mg/kg on day 4), and cyclophosphamide (60 mg/kg per day, on days 3 and 2). For patients aged 60 and over, the busulfan dose was reduced by 40%. To prevent seizures, clonazepam (0.5 mg/12 h) was administered during busulfan treatment. To prevent hemorrhagic cystitis, mesna was continuously infused during the cyclophosphamide perfusion. Patients received prophylaxis for pneumocystis and viral infections but not for fungal infections. Depending on each center’s practices, some patients received granulocyte colony-stimulating factor during the ASCT.