Bioruptor plus sonication system

The hydrodynamic diameter of aggregates of TiO₂- and CB-NPs was measured by DLS. Nanoparticles stock suspensions (2 mg ml⁻¹) were sonicated with Bioruptor^® Plus Sonication System (Diagenode Inc., Denville, NJ, USA) (20 kHz, 320 W) during 3 cycles of 30 s each. Right after the sonication, NPs were diluted as follow: in PBS 22 °C at 5, 20, 40 and 80 µg ml⁻¹ and in DMEM/F12 37 °C at 5, 10, 25 and 50 µg ml⁻¹ (these latter concentrations corresponded to exposure doses of NPs between 0.1 and 10 µg cm⁻² in cell-based experiments). NP suspensions were centrifuged for 2 s at 2000 g to remove large aggregates. The supernatants were then analyzed by DLS. Of note, the 5 µg ml⁻¹ concentration of NPs in PBS or DMEM/F12 was too low to estimate the hydrodynamic diameter by DLS. For other NPs concentrations, the hydrodynamic diameter was measured with a Vasco Kin™ particle size analyzer (Cordouan Technology, Pessac, France) in combination with the software NanoKin (V2.3.3.0). The following Vasco Kin Particle Size Analyzer parameters were chosen: temperature (22 °C or 37 °C), laser power (between 70 and 80%), acquisition mode (continuous), and analysis mode (Cumulants). The scattering angle was 170°. Each measurement was performed in triplicate (Additional files 1 and 2: Table S1 and Fig. S1).

1C11 cells were grown in Dulbecco’s Modified Eagle Medium (DMEM high glucose, GlutaMAX™ supplement, Gibco) supplemented with 10% fetal calf serum (FCS, Biochrom GmBH, Berlin, Germany). On addition of 1 mM dibutyril cyclic AMP (dbcAMP, Sigma-Aldrich, Darmstadt, Germany) and 0.05% CyclohexaneCarboxylicAcid (Sigma-Aldrich, Darmstadt, Germany), almost 100% of 1C11 cells acquire within 4 days a complete serotonergic phenotype (1C11^5−HT) [35 (link)]. Before cells were exposed to nanoparticles, nanoparticle stock solution (2 mg ml⁻¹) was sonicated with Bioruptor^® Plus Sonication System (Diagenode Inc., Denville, NJ, USA) (20 kHz, 320 W) during 3 cycles of 30 s each, then diluted to the proper concentration in serum-free DMEM/F-12 without phenol red, centrifuged for 2 s at 2000 g, and immediately used. Prepared nanoparticles were applied on cells grown to 90–95% confluency using the dose metric µg cm⁻², which refers to the concentration of nanoparticles distributed over the surface of the cell culture plate. As done with other cell paradigms [17 (link), 31 (link)], and to identify molecular pathways by which NPs promote a loss of cell homeostasis, 1C11 cells and their serotonergic neuronal progenies were acute-exposed to several concentrations of TiO₂- or CB-NPs ranging from 0.1 to 10 µg cm⁻².

HepG2 cells were grown in T75 flasks to a confluency of about 90% and transfected with 1.3× HBV genome construct. Vehicle or 10 nM calcitriol was added immediately after transfection. Cells were processed after 24 h using the EZ-ChIP Chromatin Immunoprecipitation Kit (Millipore) as per the manufacturer's protocol. Briefly, cells treated with 1% formaldehyde were washed, pelleted, and lysed to release chromatin. The cell lysate was sonicated using Bioruptor Plus Sonication System (Diagenode) for 4 × 10 cycles, for 30 s ON/30 s OFF at “high” setting to obtain DNA ranging from 200 bp to 1000 bp. The sheared chromatin was incubated with anti-VDR antibody (Abcam; ab3508), anti-RXRa antibody (Cell Signaling Technology; D6H10), or rabbit IgG (Thermo Fisher Scientific; 02-6102). Protein G Agarose beads were added to each sample the next day for 1 h at 4 °C to bind the antibody–chromatin complex, which was later eluted in the presence of 1% SDS and 0.1 M NaHCO₃. Finally, the DNA–protein crosslinking was reversed in the presence of 200 nM NaCl at 65 °C for 4 h, and the DNA was subsequently purified using spin columns provided in the kit. Real-time PCRs were performed with primers listed in Table S2 to quantitate the immunoprecipitated target DNA.