Bca assay

Cells were cultured in a 6-well plate. After treatment, the total protein of cells was obtained and quantified using a BCA assay (Sigma-Aldrich, St Louis, MO, USA). Proteins were separated according to their molecular weights through sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, MA, USA). After successive incubation with primary and rabbit secondary antibody conjugated with HRP (Abcam, Cambridge, UK), membranes were observed using enhanced chemiluminescence (Pierce, Rockford, IL, USA). Primary antibodies used were anti-GPX-1, anti-CAT, anti-SOD-1, anti-SOD-2, anti-iNOS, anti-COX-2, and anti-β-actin (1:1000; Cell Signaling Technology, Inc., Danvers, MA, USA).

The cellular uptake of ^natCu-MN-anti-miR10b was compared with that of MN-anti-miR10b and parent MN. 4T1-luc cells were seeded in a 12-well plate and incubated with ^natCu-MN-anti-miR10b, MN-anti-miR10b, and MN for 24 h at 37 °C. After washing with DPBS, the cells were lysed (Cell lysis buffer, Sigma-Aldrich, St. Louis, MO) and analyzed by ICP-MS to determine the concentration of iron. The protein concentration was determined by BCA assay (Sigma-Aldrich, St. Louis, MO). The cellular uptake of nanoparticles was normalized by total protein.

Freshly isolated mouse TBC were lysed using a micro-potter in 20 µL of TSE buffer (50 mM Tris HCl, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P40, 5 µL/mL protease inhibitors (Sigma)) [25 (link)]. Samples were stored on ice for 30 min, and then centrifuged (10,000 g, 10 min, 4°C). Lysates were used immediately or stored at −80 °C until the assay. Protein concentrations in homogenates were assayed using the BCA assay (Sigma, Saint Quentin-Fallavier, France). Denatured proteins (25 µg) were separated by SDS-PAGE (8%) and transferred to a polyvinylidene difluoride membrane. After being blocked for 3 h using a TBS buffer containing 5% BSA and 0.05% Tween-20, the membrane was incubated overnight with either of the antibodies: anti-CD36 antibody (R&D Systems, AF2519; 1:1000), anti-GPR120 antibody (Abcam, Paris, France, ab97272; 1:500), anti-α-gustducin antibody (Santa Cruz, Heidelberg, Germany, sc-395; 1:200) and anti-β-actin antibody (Santa Cruz, Heidelberg, Germany, sc-47778; 1:5000). The α-gustducin was used as an internal reference protein. After a set of washes, the appropriate peroxidase-conjugated secondary antibody was added. Antibody labeling was detected by chemiluminescence (Clarity, Bio-Rad, Marnes-la-Coquette, France).

Cells were lysed in lysis buffer (1.5% SDS, 1% NP-40, 10 mM Tris, pH 8.0, and 1 mM EDTA) containing a protease inhibitor cocktail (cOmplete™, Roche). Sonication was performed and the sample was then boiled at 95°C for 6 min. Total protein concentrations were measured using BCA assay (Sigma–Aldrich). Protein samples were separated by SDS/PAGE and transferred on to PVDF membranes. After blocking with 5% skim milk in TBS containing 0.1% Tween 20, membranes were incubated with antibodies specific for desmoglein1 (Dsg1), desmoglein3 (Dsg3), tubulin, actin (Santa Cruz Biotechnology, Santa Cruz, CA), poly(ADP-ribose) polymerase (PARP) (Cell Signaling Technology, Danvers, MA, U.S.A.), ubiquitinylated conjugates (FK2 clone, Enzo Life Sciences, lnc., Farmingdale, NY, U.S.A.), GAPDH, or Dsp (Thermo Fisher Scientific). Membranes were washed using TBS with 0.1% Tween-20 and incubated with secondary antibodies. Signals were detected using an ECL solution (Supersignal West Pico, Thermo Fisher Scientific).

Carbohydrate-specific PTS activity was assessed using cultures of S. mutans UA159, MMZ952 and ∆ccpA harvested at mid-exponential phase (OD₆₀₀=0.5). After thawing on ice, cells were washed twice in 0.1M sodium-potassium phosphate buffer (pH 7.2) containing 5mM MgCl₂ and resuspended in 10% of the initial volume using the same buffer. Cell permeabilization was achieved by vortexting with 0.05 volumes of toluene-acetone solution (1:9; vol/vol), and 50μl of the resultant suspension was used in each reaction. The 1-ml reaction mixture included 0.1M sodium potassium phosphate buffer, 5mM MgCl₂, 100μM NADH, 10mM NaF, 10mM of Glu+Fru or Suc, 10 units of a lactate dehydrogenase solution (Sigma), and 5mM PEP (0 for the blank; LeBlanc et al., 1979 (link); Moye et al., 2014 (link)). The reaction was conducted at 37°C and the rate of PEP-dependent NADH oxidation was evaluated over time by monitoring the optical density (340nm). Protein concentration was assessed using a bicinchoninic acid (BCA) assay (Sigma). Activity was expressed as nmol NADH oxidized (min)⁻¹ (mg of protein)⁻¹.

Total cellular protein was extracted using RIPA buffer (Sigma-Aldrich) and quantified with BCA assay (Sigma-Aldrich). SPC25 and PDGF were measured with ELISA kits from MyBiosource and R&D Biosystem, respectively. Western blot was performed with the following antibodies: a rabbit anti-human SPC25 (1:750; Ab236972, Abcam, Dallas, TX, USA), a mouse anti-human Egr-1 (1:2,000; Ab55160, Abcam), a rabbit anti-human PDGF (1:1,000; Ab23914, Abcam), and a mouse anti-human GAPDH antibody (1:1,000; Ab8245, Abcam). All secondary antibodies were from Jackson ImmunoResearch Labs (West Grove, PA, USA). Quantification was done with ImageJ (NIH, Bethesda, MA, USA). Immunocytochemistry was done with a mouse anti-human PDGFR alpha antibody (1:50; Ab96569, Abcam).

Cell total protein was extracted using RIPA buffer supplemented with phosphatase and protease inhibitors (Roche Diagnostics, Meylan, France). Protein concentration was determined using a BCA assay (Sigma Aldrich) and equal amounts of protein were resolved by SDS-PAGE. The blocked membrane was incubated with primary antibodies overnight at 4 °C. Bands were detected by using a horseradish peroxidase secondary antibody and visualized with ECL Plus reagent (GE Healthcare, Buc, France). β-Tubulin was used as loading control (Santa Cruz Biotechnology, Heidelberg, Germany).