Figure 2. Ceapin-induced foci are reversible and correlate with inhibition of ATF6.

(A–C) Time-lapse images of U2-OS cells stably expressing GFP-ATF6α treated either with vehicle (A, DMSO) or active Ceapin analogs (B, 10 μM Ceapin-A1), (C, 1 μM Ceapin-A7) for seventeen minutes to allow foci formation. Cells were then washed once with PBS and then media without compound was added. Scale bar is 10 μm. Images are representative of three independent experiments where three positions per well were imaged for each experiment. (D–G) Time-lapse images of U2-OS cells stably expressing GFP-ATF6α treated either with vehicle (A,B, DMSO) or 10 μM Ceapin-A1 (C,D). After fifteen minutes, the cells were washed with PBS and then media without (A,C, DMSO) or with ER stressor (B,D, 100 nM Tg) was added. After washout, ATF6α inhibitor foci resolve (C,D) and nuclear translocation of GFP-ATF6α occurs with similar kinetics in cells initially treated with either DMSO (B) or Ceapin-A1 (D). Scale bar is 10 μm. Images are representative of three independent experiments where three positions per well were imaged for each experiment.

DOI: http://dx.doi.org/10.7554/eLife.11880.013

Figure 2—figure supplement 1. Redistribution of GFP-ATF6 from foci into the ER after washout of Ceapin-A1 does not require protein synthesis.

(A–D) Time-lapse images of U2-OS cells stably expressing GFP-ATF6α pretreated with either cycloheximide (A, B 0.1 μg/ml CHX) or vehicle (C, D ethanol) for three hours prior to imaging. During imaging, cells were treated either with vehicle (A, C DMSO) or Ceapin-A1 (B, D 10 μM Ceapin-A1) for sixteen minutes to allow foci formation. Cells were then washed once with PBS with (A, B) or without (C, D) cycloheximide and then media without compound with (A, B) or without (C, D) cycloheximide was added. Scale bar is 10 μm. Images are representative of three independent experiments where three positions per well were imaged for each experiment.

DOI: http://dx.doi.org/10.7554/eLife.11880.014