Fig 1.
Low temperature restores apical membrane localization of particular ABCB4 mutants.
(A) Representative double immunofluorescence staining for ABCB4 (red) and the ER marker calnexin (green) in MDCK-II cells expressing wild-type ABCB4 after incubation at 37°C or 30°C. Areas of yellow in the merged images indicate colocalization of ABCB4 and calnexin. Optical horizontal x,y (upper) and vertical x,z sections (lower) are shown. (B) Confocal microscopy images of cells expressing ABCB4 mutants. For simplicity, only the merged images are shown. Reducing temperature improved cell surface localization for the mutants G228R and A934T, as revealed by the appearance of red signal around the apical membrane. All images represent three independent transfections. Bars, 20 μm.
Fig 2.
Chemical chaperones rescue cell surface expression of the ABCB4 mutants G228R and A934T.
Transfected MDCK-II cells were treated with 0.1% DMSO (vehicle), 1 mM 4-PBA or 1 μM curcumin during 24 h. The cells were double stained for ABCB4 and calnexin and analyzed by confocal microscopy. The merged images of ABCB4 and calnexin are presented. Co-localization (yellow) of calnexin with G68R or D459H mutants was seen after addition of any of the chaperone drugs. Positive apical staining for ABCB4 (red) was found in cells expressing the G228R and A934T mutants following treatment with either 4-PBA or curcumin. The images are representative of four independent experiments, in which at least two different batches of each plasmid were used. Bars, 20 μm.
Fig 3.
Effect of chemical chaperones on the processing of mutant ABCB4 proteins.
(A) Western blots with anti-ABCB4 (top) and anti-Na/K-ATPase (bottom) antibodies of cell lysates (30 μg) from AD-293 transfected cells untreated or treated with 1 mM 4-PBA or 1 μM curcumin. The images are representative of four independent experiments. Numbers below the blots represent the mean±SD of the ratio between mature and immature forms of ABCB4, as determined by densitometric quantification of the signals. (B) Real-time quantitative PCR analysis. ABCB4 mRNA measurements were normalized to β-actin. Values represent the mean±SD of four independent experiments and are expressed relative to untreated cells (Control).
Fig 4.
Effects of 4-PBA on the stability and maturation of the G228R and A934T mutants.
(A) AD-293 cells expressing wild-type ABCB4 or the G228R or A934T mutants were incubated with cycloheximide (50 μg/ml) for 8 or 16 h in the presence or absence of 1mM 4-PBA. Equal amounts of proteins (30 μg) were fractionated by 6% SDS-PAGE and subjected to western blotting. (B) Transfected cells were untreated (control) or treated for 2 h with BFA (1 μg/ml). BFA-treated cells were subsequently rinsed and further incubated for 8 h with or without 1 mM 4-PBA. The total amount of protein loaded onto each lane was 30 μg. The images are representative of three independent experiments.
Fig 5.
Chemical chaperones rescue phospholipid efflux activity of the ABCB4 mutants G228R and A934T.
Transfected AD-293 cells were labeled with 2 μCi/mL of [3H]-choline and incubated for 3 h in EMEM with or without 1mM NaTC, as indicated. Aliquots of the culture medium were harvested each 30 min and the radioactivity released was measured by liquid scintillation counting. Values were normalized to the levels of ABCB4 mRNA and are expressed relative to total cell protein. (A and B) Time course of [3H] release in the presence or absence of NaTC in cells expressing wild-type ABCB4 (A) or ABCB4 mutants (B). (C) NaTC-dependent efflux of radioactivity in cells expressing wild-type ABCB4 previously treated for 24 h with 0.1% DMSO (control), 1 mM 4-PBA, or 1 μM curcumin. (D) NaTC-dependent efflux of radioactivity in cells expressing ABCB4 mutants after treatment with 1 mM 4-PBA or 1 μM curcumin; H2O and 0.1% DMSO were used as vehicle, respectively. A representative experiment out of four is shown. For cells expressing the mutants G228R and A934T, differences in NaTC-dependent radioactivity efflux rate between vehicle- and 4-PBA- or curcumin-treated cells were statistically significant (p < .05).