Volume 131, Issue 3, Pages 878-884 (September 2006) Radixin Is Required to Maintain Apical Canalicular Membrane Structure and Function in Rat Hepatocytes  Wei Wang, Carol J. Soroka, Albert Mennone, Christoph Rahner, Kathy Harry, Marc Pypaert, James L. Boyer  Gastroenterology  Volume 131, Issue 3, Pages 878-884 (September 2006) DOI: 10.1053/j.gastro.2006.06.013 Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions

Figure 1 Adenovirus-mediated siRNA effectively inhibited radixin expression in collagen sandwich–cultured rat hepatocytes. (A) Clone 9 cells were treated with PBS, Ad-siControl, or the 4 siRNAs individually or in various combinations at a virus dose of multiplicity of infection 10. The cells were harvested 3 days after treatment and cell lysates were separated by polyacrylamide gel electrophoresis, and blotted for radixin and normalized to β-actin. (B) Rat hepatocytes treated with PBS, Ad-siControl, or Ad-siRDXC were harvested 1, 3, and 5 days after the treatment and cell lysates were blotted for radixin and β-actin. (C) Hepatocytes were treated with PBS, Ad-siControl, or Ad-siRDXC with multiplicity of infection 25 or 50, harvested on day 5, and cell lysates were blotted for radixin and β-actin. (D) The hepatocyte lysates also were blotted for Mrp2, Bsep, Mrp3, and Mdr1. A representative result of at least 3 experiments is shown. Gastroenterology 2006 131, 878-884DOI: (10.1053/j.gastro.2006.06.013) Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions

Figure 2 Ad-siRadixin reduced canalicular structures in sandwich-cultured hepatocytes. (A) Differential interference contrast images of hepatocytes that were treated with PBS, Ad-siControl, or Ad-siRadixin were acquired on days 1, 3, and 5. Five-day cultures of cells treated with PBS, Ad-siControl, or Ad-siRadixin were labeled with (B) anti-radixin antibody or (C) Alexa 594 phalloidin for actin filaments. Both show loss of apical canalicular structures after Ad-siRadixin treatment. Bar, 10 μm. Gastroenterology 2006 131, 878-884DOI: (10.1053/j.gastro.2006.06.013) Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions

Figure 3 Electron micrographs of sandwich-cultured hepatocytes. Hepatocytes were treated with PBS, Ad-siControl, or Ad-siRadixin and cultured for 5 days. Hepatocytes treated with (A) PBS or (B) Ad-siControl developed dilated bile canaliculi. Tight junctions are intact and few microvilli are observed. (C) The bile canalicular lumina of Ad-siRadixin–treated cells were largely collapsed. The tight junctions remain intact and the loss of severe dilation results in the appearance of microvilli. BC, bile canaliculi. Bar, 2 μm. Gastroenterology 2006 131, 878-884DOI: (10.1053/j.gastro.2006.06.013) Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions

Figure 4 Ad-siRadixin altered the localization of apical bile transporters in hepatocytes. Hepatocytes were treated with PBS, Ad-siControl, or Ad-siRadixin and cultured for 5 days. Cells were double-labeled with anti-Mrp2 and anti-Bsep antibodies (top 2 rows), with anti-Mdr1 (third row), or anti-Oatp2 antibodies (bottom row). Note that all 3 apical membrane transporters, but not the basolateral membrane protein, showed significant intracellular localization in knock-down areas after Ad-siRadixin treatment. Arrow shows collapsed canaliculi. Bar, 10 μm. Gastroenterology 2006 131, 878-884DOI: (10.1053/j.gastro.2006.06.013) Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions

Figure 5 The intracellular fraction of Mrp2 was colocalized with Rab11. In Ad-siControl cells Mrp2 (green) was localized to the dilated bile canaliculi. Occasional weak staining was seen intracellularly. Rab11 (red) was found in a subapical and perinuclear compartment in these cells. The merged image shows the separation of these compartments. In contrast, in Ad-siRadixin–treated cells, knock-down areas (as defined by the lack of intact, dilated bile canaliculi) revealed extensive perinuclear colocalization of both Mrp2 and Rab11. Occasional, stubby canaliculi were seen labeled for Mrp2 (arrow). Bar, 10 μm (top 2 rows). High magnification of the knock-down area showed that Mrp2 was largely colocalized with Rab11 in the intracellular compartment. Bar, 5 μm (bottom row). Gastroenterology 2006 131, 878-884DOI: (10.1053/j.gastro.2006.06.013) Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions

Figure 6 Ad-siRadixin decreased the rate of biliary excretion of glutathione-methylfluorescein and CGamF. Hepatocytes were treated with PBS, Ad-siControl, or Ad-siRadixin and cultured for 5 days. (A) Cells were incubated with CMFDA or CGamF and visualized by confocal microscopy. Bar, 10 μm. (B) Hepatocytes were treated with standard or Ca2+-free HEPES buffer before the bile excretion assay. The Biliary Excretion Index was measured as the percentage of glutathione-methylfluorescein fluorescence released into canalicular lumina. Data represent the means ± SD of 5 experiments. P > .05, PBS–compared with Ad-siControl–treated cells. *P < .05, Ad-siRadixin–compared with PBS- or Ad-siControl–treated cells. Gastroenterology 2006 131, 878-884DOI: (10.1053/j.gastro.2006.06.013) Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions