Figure 2. The ER remains connected and distribution of CPY*-mRFP, GFP-CFTR, and Ire1-GFP aggregates is similar between the cER and pnER in unstressed and ER stressed cells.
(A and B) Fluorescence loss in photobleaching (FLIP) analysis of Kar2-sfGFP-expressing WT cells incubated with DMSO or 1 μg/ml Tm for 30 min. A region in the cER was photobleached (black rectangle) and the loss of fluorescence in the cER (orange rectangle) or pnER (green rectangle) was monitored. Average fluorescence depletion curves normalized to the pre-bleach fluorescence intensity are shown. Graphs are the mean ± SD of 3 experiments, each examining ≥5 cells.
(C) WT cells expressing the Hmg1-GFP ER reporter were grown in synthetic media with 2% galactose for 2 hr (with DMSO or 1 μg/ml Tm) to induce CPY*-mRFP expression.
(D) WT cells expressing the ER reporter DsRed-HDEL were incubated with 100 μM copper sulfate for 2 hr (with DMSO or 1 μg/ml Tm) to induce GFP-CFTR expression.
(E) WT cells expressing the DsRed-HDEL ER reporter and Ire1-GFP were treated with DMSO or Tm (1 μg/ml) for 1 or 3 hr.
In all experiments, foci were quantified and shown by the ratio of foci in the pnER to that in cER per 50 μm2 surface area (SA) and are the mean ± SD of 3 experiments, each examining ≥100 cells. Scale bar is 2 μm.