Nis elements ar 4

Morphological changes of the cells (3T3 and HaCaT) were evaluated via laser scanning confocal microscopy. After 48-h incubation of cells in the same way as in the toxicity studies, using the enhancer concentration corresponding to their IC₁₅ and IC₈₅, cell samples were prepared for confocal microscopy (see Supplementary data for details). Finally, eight focal planes (pinhole diameter = 19.16 μm) were taken of each sample using a Nikon A1 + confocal system (Nikon, Tokyo, Japan) equipped with NIS Elements AR 4.20 software (Laboratory Imaging, Prague, Czech Republic).

COS-1 cells were seeded on a 48-well plates (80,000 cells/well) and were incubated for 24 h. The cells were transfected (Lipofectamine^® 3000) with 100 ng/well pEGFP-hCAR+Ala construct. The cells were treated 24 h after transfection with CITCO (10 μM), erlotinib (10 μM), phenobarbital (500 μM), LEF, and TER (30 μM), or vehicle (0.1% DMSO). For final counting cells were stained with Hoechst 33342 (10 μg/ml, for 5 min in CO₂ incubator). Confocal microscopy was performed at 0, 24, and 48 h after treatment with a Nicon Ti microscope and Nikon A1 plus camera (Nikon, Japan) using 405 and 488 nm lasers. The pinhole diameter was set at 35.76 μM and microphotographs were taken using NIS Elements AR 4.20 software (Laboratory Imaging, Czechia). Four photographs of every treatment were taken and cytoplasmic, nuclear and mixed localization was determined in at least 100 cells in three independent experiments (n = 3). pEGFP-hCAR construct kindly donated by Dr. Y. Kanno (Kanno et al., 2007 (link)) was used for site directed mutagenesis (GeneArt^TM Site-Directed Mutagenesis System, Thermo Fisher Scientific, Waltham, MA, United States) to insert extra alanine at position 271 of the hCAR LBD with primers 5′-CCCTCTTCTCTCCTGCTGACCGACCTGGAGTTAC-3′ and 5′-GTAACTCCAGGTCGGTCAGCAGGAGAGAAGAGGG-3′ as previously described (Chen et al., 2010 (link)).

The cells were seeded in 4-well cell imaging slides (4 × 10⁵ cells/well). After 48 h incubation cells were fixed using a solution of 4% paraformaldehyde in PBS (15 min) at room temperature (RT). Cells were treated with BSA blocking buffer (5% in PBS) for 90 min at RT, then the cells were incubated on ice with Megalin polyclonal antibody (0,01 mg/mL, 4 h) followed by incubation with secondary anti-rabbit CF™ 594 antibody (5 µg/mL, 1 h). In the next step, the cells were stained with Hoechst 33342 (1 µg/mL, 30 min, ThermoFisher Scientific) to visualize nuclei. To stain the actin filaments ActinGreen 488 ReadyProbes reagent (ThermoFisher Scientific) was used according to the manufacturer’s instructions. Excess salts were removed by washing with demineralized water (5 min). Dry samples were mounted with ProLong Gold antifade reagent (Invitrogen), dried, nail-polished and analyzed by Nikon A1 + confocal system (Nikon) equipped with NIS Elements AR 4.20 software (Laboratory Imaging).