treptomycin. Adherent cells were then MedChemExpress Tangeritin incubated with AlexaFluor488-labelled fetuin-A-containing synthetic CPP in complete culture medium for 30 minutes at 37uC. In order to differentiate between cell-bound and internalized CPP, rabbit anti-AlexaFluor488 IgG was used to quench cell-surface fluorescence. At given time points, cells were washed with ice-cold PBS and incubated in PBS as control, or 80 mg/mL anti-AlexaFluor488 IgG in PBS for 1 h at 4uC. Cells were Fetuin A Calciproteins in Macrophage 6 Fetuin A Calciproteins in Macrophage then fixed in 4% paraformaldehyde in PBS for 15 min at RT and mounted in ProLong Gold Antifade Reagent. 19374401 Images were captured using a Fluoview FV1000-IX81 confocal microscope. To study CPP internalisation kinetics, RAW 264.7 cells were seeded on 96-well plates at a density of 5000 cells per well in DMEM culture medium supplemented with 10% FBS, 2 mmol/L L-glutamine for 12 h at 37uC. Adherent cells 
were then incubated with AlexaFluor488-labelled fetuin-A-containing synthetic CPP, and then subsequently with quenching antibody or PBS alone as described above. The efficiency of cell-surface quenching with anti-AlexaFluor488 IgG was estimated by measuring the fluorescence after incubation of cells with labeled CPP at 4uC, as CPP uptake should be minimal at this temperature. Unquenchable fluorescence was determined at each time point and accounted for 5 to 15% of the total cell-associated signal. Internalization of CPP was calculated using the ratio of quenched signal to unquenched signal after correction for the unquenchable fluorescence as described by Gostring et al. Total cell associated fluorescence was measured using a Synergy HT multimode plate reader with appropriate excitation/emission filters. Given that FBS contains large amounts of bovine fetuin, serumfree medium was used to culture cells for the assessment of monomeric fetuin-A uptake. Cells were washed and cultured in serum-free medium for 2 h prior to treatment with CPP or HAP-containing medium containing purified human fetuin-A at the concentrations indicated. To block apoptosis, cells were pre-incubated with 20 mmol/L zVAD-fmk for 1 h before treatment with CPP/HAP. To block endocytosis via SR-AI, cells were pre-incubated with rat monoclonal anti-mouse SR-AI blocking antibody or rat IgG2b isotype control for 1 h prior to addition of CPP or HAP-containing culture medium for a further 24 h. For assessment of macrophage proliferation, RAW cells were plated in 24-well plates at 10000 cells per well and left overnight in culture medium. After attachment, the medium was changed to DMEM supplemented with only 1.0% FBS for 48 h to induce quiescence. This medium was then replaced with growth medium with or without CPP/HAP, at the stated concentrations, for 48 h. To quantitate the number of cells in culture, the medium was aspirated and the cells gently scraped. Viable cells were 11121575 identified by trypan blue exclusion and were counted in a hemocytometer. For the detection of apoptosis, cells were plated in 96-well plates as for MMT assay, and then incubated in culture medium containing CPP/HAP at the stated concentrations for 24 h. After fixation and permeabilisation of the cells, plates were processed for a TUNEL based assay using the Click-iT TUNEL Alexa Fluor 488 Imaging Assay. DNase I-treated cells were used as a control. To confirm the findings by TUNEL assay, replica plates were fixed with 80% methanol in PBS and then processed for assay using the ssDNA A