A1 plus camera

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)).

COS-1 cells were seeded on 48-well plates (20,000 cells/well) and, 24 h after seeding, were transfected (Lipofectamine™ 3000) with 100 ng/well pEGFP-hCAR+Ala construct. The cells were treated with CITCO (10 μM), diazepam, nordazepam, temazepam and oxazepam (10 μM and 30 μM), or control (DMSO 0.1%) for 24 h. For microscopy, living cells were stained with Hoechst 33342 (0.2 µM, 5 min at 37 °C) (Sigma-Aldrich). Confocal microscopy was performed with a Nicon Ti ECLIPSE microscope and Nikon A1 plus camera (Nikon, Tokyo, Japan) using 405 and 488 nm lasers. Microphotographs were taken using the NIS Elements AR 4.20 software (Laboratory Imaging, Czech Republic). Four microphotographs of every treatment were taken and cells with green fluorescence protein (GFP) cytosolic or nuclear localization was counted in more than 150 cells in each picture. Each experiment was performed in biological triplicates (n = 3).
pEGFP-hCAR construct kindly donated by Dr. Y. Kanno [21 (link)] was used for site directed mutagenesis (using GeneArtTM Site-Directed Mutagenesis System, Thermo Fisher Scientific, Waltham, MA, USA). To insert extra alanine at position 271 of the CAR-LBD, the primers were used as previously described (50-CCCTCTTCTCTCCTGCTGACCGACCTGGAGTTAC-30 and 50-GTAACTCCAGGTCGGTCAGCAGGAGAGAAGAGGG-30) [18 (link)].

For laser scattering confocal microscopy, BMM were seeded onto confocal dishes (SPL, glass bottom, 101350, SPL Life Sciences, Gyeonggi-do, Korea) in a density of 100,000 cells/cm². After overnight stabilization, cells were treated for 1 h with vehicle (DMEM), RhB solution (15 µg/mL) or with RhB-loaded nanosphere samples (300 μg/mL). Living cell nuclei were stained with Hoechst 33,342 (0.2 µM, 5 min at 37 °C). For microscopy Nicon Ti ECLIPSE microscope and Nikon A1 plus camera (Nikon Instruments, Melville, NY, USA) using 405 and 561 nm lasers was used. The pinhole diameter was set to 19.16 µM and microphotographs were taken using the NIS Elements AR 4.20 software (Laboratory Imaging, Prague, Czech Republic). Three representative photographs of every treatment were taken.