Fig. 4

CHMP5 regulates the apical targeting of BSEP and BSEP-mediated bile acid secretion. a Representative images demonstrate the impaired membrane targeting of BSEP in CHMP5-knockdown Hep G2 cells. Hep G2 cells were pretreated with CHMP5-targeting (si-CHMP5) or non-targeting (si-CTL) siRNA pools, and then co-expressed FLAG-BSEP and the membrane marker Mem-DsRed for temperature shift assays. Cell nuclei, FLAG-BSEP and Mem-DsRed were stained by DAPI, anti-FLAG and anti-mCherry antibodies, respectively. For clarity, the squared images are enlarged versions of the white open squares; arrows indicate the FLAG-BSEP on the plasma membrane. The bar graph (n = 4, mean ± SD) illustrates the subcellular distribution index, in which total 121 and 96 cells in the si-CTL and the si-CHMP5 groups, respectively, were analyzed. Two-tailed unpaired Student’s t-test was used (*P = 0.0286). b Representative images reveal defective apical targeting of BSEP in Chmp5-knockdown mouse livers. The plasmid pool expressing shRNA targeting mouse Chmp5 (sh-Chmp5) or the scramble sequence (sh-CTL) and p3XFLAG-BSEP were hydrodynamically injected into FVB/NJ mouse livers. The distribution of FLAG-BSEP (green) in these liver samples were immunostained by anti-FLAG antibodies. The arrow indicates the accumulated subapical BSEP. The bar graph (n = 3, mean ± SD) illustrates the canalicular distribution of FLAG- BSEP in Chmp5-knockdown mouse livers. More than 10 images per mouse and three mice in each group were analyzed for the canalicular targeting index. c The bar graph (n = 4; mean ± SD) illustrates that BSEP-mediated bile acid secretion was reduced in CHMP5-knockdown cells and rescued through overexpressing CHMP5. The total bile acids concentrations were determined from the conditional medium of Hep G2 cells with indicated transfection. The plasmid pmCherry-C1 was used as a negative control for CHMP5 rescue. This experiment was performed in quadruplicate. Two-tailed unpaired Student’s t-test was used (*P ≤ 0.05). The P-value is 0.0314 and 0.0402 of the left and the right analysis, respectively