Ab190797

HUH7 human hepatoma cells were incubated for 24 h in medium containing BSA. To see whether AN1284 can reduce steatosis from an FFA by a direct action on liver cells, PA was added together with different concentrations of AN1284 for 24 h. Lipid content was quantified by ORO staining. Since RNA-seq analysis suggested that AN1284 could act via the aryl hydrocarbon receptor (AhR), we measured the effect of AN1284 on the nuclear translocation of AhR, by immunofluorescence intensity, 15 min after its addition to the cells. We used a specific antibody (Abcam, ab190797), analyzed its intensity with ImageJ, and normalized it to the control group. RT-qPCR was used to measure the target genes of AhR after 24 h: fatty acid synthase (FASN), SREBP-1c, acyl-CoA oxidase 1 (ACOX1), and cytochrome P450-1 (CYP1a1). siRNA for human AhR from TriFECTa Kit DsiRNA Duplex purchased from ITD was used to silence AhR. The reverse transfection of these siRNAs onto HUH7 cells was performed by means of TransIT-X2 Dynamic Delivery System (MC-MIR-6000, Mirus) according to the manufacturer’s instructions. To examine the effect of siRNA on AhR expression, total protein was extracted from cells 48–72 h after transfection, and AhR protein levels were measured using Western blot (WB) with primary AhR antibody (Abcam, ab190797). The siRNA-transfected cells were treated with AN1284 and analyzed.

Briefly, lysates from cells and tumor tissues were prepared, and protein levels determined using the BCA assay (Bio-Rad, Hercules, CA). A total of 30 μg protein from each sample was resolved by SDS–PAGE on Tris-glycine gels and transferred to PVDF membrane. Membranes were blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline containing 0.1% Tween 20 (TBST) and incubated with primary antibodies overnight at 4 °C. Membranes were washed three times for 5 min each with TBST, incubated in either HRP-goat-anti-mouse (ab6789, Abcam) or HRP-goat-anti-rabbit (ab6721, Abcam) secondary antibodies for 2 h at room temperature. Immunoreactive bands were visualized using Pierce ECL plus Western blotting substrate (32132, Thermo Fisher Scientifc). The primary antibodies used in the present study were against: AR (5153 S, Cell Signaling Technology, Boston, MA, USA), PSA (5365 S, Cell Signaling Technology), β-actin (4970 S, Cell Signaling Technology), c-Caspase-3 (9661 S, Cell Signaling Technology), Bax (2774 S, Cell Signaling Technology), Bcl-2 (15071 S, Cell Signaling Technology), ubiquitin (3936 S, Cell Signaling Technology), and AHR (ab190797, Abcam). The protein bands were analyzed using ImageQuant 5.2 software. The expression of β-actin was used as a loading control.