Sodium 4 pba

The generation of the ABCB4 mutants G68R, G228R, D459H and A934T has been previously described [13 (link),16 (link)]. Madin-Darby canine kidney MDCK-II cells and human embryonic kidney AD-293 cells were obtained from ATCC (LGC Standards Barcelona, Spain) and Agilent Technologies (Santa Clara, CA, USA), respectively. The mouse monoclonal anti-ABCB4 antibody (clone P3II-26) was purchased from Millipore (Billerica, Masachussets, USA). The rabbit anti-calnexin antibody was obtained from StressMarq (Victoria, Canada), and the anti-Na/K-ATPase antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-mouse AlexaFluor594-conjugated and anti-rabbit AlexaFluor488-conjugated secondary antibodies were from Molecular Probes (Eugene, OR, USA). Sodium 4-PBA, curcumin, cycloheximide, brefeldin A and standard lipids (phosphatidic acid, phosphatidylinositol, phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, sphingomyelin) were provided by Sigma (St. Louis, MO, USA). [³H]-choline (60–90 Ci/mmol) was purchased from Perkin Elmer (Massachusetts, USA). All other reagents were of analytical grade.

A Senescence β-Galactosidase Staining Kit (Cell Signaling Technology, Inc., Danvers, MA, USA) was used to assess cellular senescence, in accordance with the manufacturer’s instructions. Nuclei were counterstained with Hoechst. SA-β-gal-positive cells and total cell numbers in a field of view were automatically counted by an IN Cell Analyzer 6000, and the positive ratio was calculated. For assessing the effects of different chemical compounds on cellular senescence, 3×10⁴ cells were seeded on day 0, cultured with each compound for two weeks, and the SA-β-gal-positive cell ratio was then calculated. The compounds used in these analyses included 2 mM sodium 4-PBA, (Sigma-Aldrich, St. Louis, MO, USA), and 2.5 mM N-Acetyl-L-cysteine (NAC, Sigma-Aldrich).

A PROTEOSTAT Aggresome Detection kit (Enzo Life Sciences Inc.) was used to detect aggresomes in cells, in accordance with the manufacturer’s instructions. Nuclei were counterstained with Hoechst. Cytoplasm was stained with HCS CellMask Deep Red Stain (Thermo Fisher Scientific Inc.). All images were collected with an IN Cell Analyzer 6000. To assess the effects of different chemical compounds on aggregate accumulation, cells were cultured with each compound and aggregate intensities were evaluated. The compounds used in these analyses included trimethylamine N-oxide (TMAO, Sigma-Aldrich), sodium 4-PBA, Sigma-Aldrich), geldanamycin (TCI, Tokyo, Japan), 2-hydroxypropyl-β-cyclodextrin (Sigma-Aldrich), rapamycin (Sigma-Aldrich), and valproic Acid (VPA, FUJIFILM Wako Pure Chemical Corporation).