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. 2016 Jan 25;63(6):1842–1859. doi: 10.1002/hep.28398

Figure 2.

Figure 2

Inhibitors of p38 and JNK correct localization and trafficking of ATP7BH1069Q mutant. (A) HeLa cells were infected with Ad‐ATP7BWT‐GFP or Ad‐ATP7BH1069Q‐GFP, incubated overnight with 200 μM BCS, and fixed or incubated for an additional 2 hours with 100 μM CuSO4. In response to Cu, ATP7BWT (left column) traffics from the Golgi (arrowheads in upper panel) to the PM and vesicle (arrows in lower panel), while ATP7BH1069Q (second column) is retained within the ER under both low‐Cu and high‐Cu conditions. The p38 inhibitor SB202190 (5 µM) or the JNK inhibitor SP600125 (2 µM) was added to the cells 24 hours before fixation (as indicated in the corresponding panels). Fixed cells were further labeled for TGN46 and visualized under a confocal microscope. Both p38 and JNK inhibitors corrected ATP7BH1069Q from the ER to the Golgi (arrowheads in upper panels) under low‐Cu conditions and to the PM and vesicles (arrows in lower panels) in high‐Cu conditions. (B) Cells were treated with BCS and drugs as shown in (A). Fluorescence of ATP7B signal in the Golgi (average ± standard deviation, n = 40 cells) was quantified and normalized to total cell fluorescence. Both inhibitors caused an increase of ATP7BH1069Q signal in the Golgi region. (C) Cells were treated as in (A). The percentage of cells (average ± standard deviation, n = 10 fields) with an ATP7B signal in the ER, Golgi, or PM/vesicles was calculated. Both p38 and JNK inhibitors reduced the percentage of cells exhibiting ATP7BH1069Q in the ER and increased the number of cells in which ATP7B was corrected to the Golgi under low‐Cu conditions and to the PM and vesicles upon Cu stimulation. (D) Cells were treated as in (A) and prepared for immuno‐EM (see Materials and Methods). Arrows indicate ATP7BH1069Q in the ER. Arrowheads show ATP7BWT or ATP7BH1069Q in the Golgi. Empty arrows indicate ATP7BWT or ATP7BH1069Q signal at the PM, while asterisks label ATP7B‐positive multivesicular body‐like vesicles. Scale bars = 5 μm (A) and 260 nm (D). Abbreviations: inh, inhibitor; WT, wild type.