Transit lt1

Dual luciferase reporter gene assays for CAR, PXR and FXR were conducted to analyze the capability of Pi and Te to activate these nuclear receptors in HepG2 cells. The plasmids and assays have been described in detail before [16 (link)]. In brief, HepG2 cells were cultivated in 96-well plates and transiently transfected with plasmids (Supplementary Table S5) using TransIT-LT1 (Mirus Bio LLC, Madison, WI, USA) in a relation of 3:1 (TransIT-LT1 [μL]: amount of plasmids [μg]). For the FXR transactivation assay, the first plasmid is based on a fusion protein of GAL4 with the ligand-binding domain (LBD) of FXR. The second plasmid contains a firefly luciferase reporter gene under control of the GAL4-specific upstream activation sequence (UAS). For the CYP7A1 promoter assay, the luciferase reporter construct is driven by a fragment of the promoter of the human FXR-responsive CYP7A1 gene [14 (link)]. All measurements were performed according to the Dual Luciferase Assay protocol as provided by the manufacturer (Promega, Madison, WI, USA) and detailed elsewhere [19 (link),20 (link)] by using a plate reader (Infinite M200PRO, Tecan, Männedorf, Switzerland).

BSR-T7/5 cells in 6-well plates with 70–80% confluence were transfected with the following components: pAmp-TCRV Sseg-cRNA (1000 ng), pAmp-TCRV Lseg-cRNA (1000 ng), pCAGGS-T7 (250 ng), pCAGGS-L (1000 ng), pCAGGS-NP (250 ng). The transfection was performed with TransIT-LT1 (Mirus Bio) according to the manufacturer’s instructions using 5 μl TransIT-LT1/μg DNA. One day after transfection medium was exchanged against fresh MEM (Glasgow) + 2% NCS. Seven days post-transfection Vero76 cells seeded for 70–80% confluence in 6-well plates were infected with 1ml of supernatant from the transfected cells for 1h, after which DMEM + 2% FCS was added without removing the inoculum. CPE development on Vero76 cells was monitored and once significant CPE was observed, a virus stock was grown. Here a T162 flask of 70–80% confluent Vero76 cells was infected with a 1:10 dilution of virus supernatant for 1 h, after which the inoculum was removed and DMEM + 2% FCS added. The cells were incubated for 5–7 days and CPE development was monitored until approximately half of the cells were rounded up, at which point the supernatant was collected and cleared of cell debris by centrifugation for 15 min at 800 x g. An additional 5% FCS was then added and the virus was aliquoted for storage at -80°C. All rTCRV stocks were titrated by plaque assay and the full genome sequences were determined as described below.

293T cells were cultured in DMEM supplemented with 10% FBS. The cells (40,000 cells) in 96-well plates were transfected by using TransIT-LT1 (Mirus Bio, WI, USA) with various amounts of expression plasmid (total amount was adjusted to 250 ng by adding empty vector pcDNA3.1), and 5 ng of firefly luciferase reporter plasmid. The cell lysates were prepared 46 hr after the transfection and subjected to dual-luciferase assay by luminometry (Promega, WI, USA).

One day prior to transfection cells were seeded and let grow into 6-well plates containing sterile glass coverslips. Respectively 1 × 10⁶ and 5 × 10⁵ S2R⁺ and HepG2 cells were transfected with 1 μg of purified plasmids DNA using TransIt LT1 (Mirus Bio, Madison, WI, USA).
For immunofluorescence staining, the cells attached to slides were washed with phosphate-buffered saline and fixed with 4% formaldehyde for 10 minutes at room temperature followed by three washes in PBS. Blocking was performed with a solution containing 10% fetal bovine serum and 0.5% of Triton X-100 for 30 minutes followed by two washes in PBS for 2 minutes each.
Cells were incubated with a dilution 1:500 of V5 antibody (Invitrogen, Carlsbad, CA, USA) conjugated with fluorescein isothiocyanate (FITC) fluorochrome for 2 hours. After three washes in PBS, the cells were stained with DAPI (4',6-diamidino-2-phenylindole) and mounted with anti-fade 1,4-diazabicyclo[2.2.2]octane (DABCO).
Slides were imaged under an Olympus (Tokyo, Japan) epifluorescence microscope equipped with a cooled CCD camera. At least 100 positive cells per slide were observed. Grey-scale images, obtained by separately recording FITC and DAPI fluorescence, were pseudo-colored and merged to obtain the final image using Adobe Photoshop program.

COS-7 cells (purchased from ATCC: CRL-1651) and NIH-3T3 cells (purchased from ATCC: CRL-1658) were cultured in DMEM (Gibco) supplemented with 10% Fetal Clone III (Hyclone) and 1% GlutaMAX (Gibco) and transiently transfected with Trans-IT LT1 (Mirus). NIH-3T3 cells were transferred to serum-free medium immediately prior to transfection to accumulate cells in G1 for visualization of the primary cilium.