Anticar

The effect of DEX on the expression and nuclear localization of CAR were evaluated in vitro in a human cell line of hepatoblastoma cells (HepG2), according to the protocol described in detail in Castellani et al. [29 (link)]. Briefly, HepG2 cells were seeded in a 24-well plate with glass coverslips. After 24 hours, cells were pre-treated either with 0.2 or 2 μM DEX for 4 hours and then with 1 ng/mL LPS for 24 hours. The effect of DEX was evaluated using untreated and LPS-treated cells as negative and positive controls, respectively. At the end of the treatment, cells were fixed in 4% paraformaldehyde and incubated with a rabbit polyclonal primary antibody antiCAR (1:100 dilution, Abcam, Cambridge, UK) for 1 h, using an Alexa Fluor 488 anti-rabbit as secondary antibody (1:500 dilution, Jackson ImmunoResearch Europe Ltd., Ely, UK). For nuclear staining, cells were incubated with DAPI (100 pg/mL, Life Technologies, Monza, Italy), after treatment with a 2 mg/mL RNAse solution (Sigma-Aldrich, Milan, Italy). The images of the immunostained cells were acquired by means of a confocal microscope Zeiss LSM 800, using a 63X magnification. ImageJ software was used to quantify the intensity of the fluorescent signal and the co-localization between the nuclear marker DAPI and CAR signal.