(A) HEK293 CRISPRi cells stably expressing doxycycline inducible 3xFLAG-ATF6α with ABCD3 or NegCtrl KD were treated either with DMSO or Ceapin (6 μM) for 30 min prior to fixation, staining with anti-ABCD3 and/or anti-PEX14, and confocal fluorescent imaging. Scale bar, 10 μm. Images are representative of two independent experiments, in which we imaged at least 20 positions per well for each experiment. (B and C) Plotted is the mean and standard deviation of the mean per cell correlation of 3xFLAG-ATF6α and ABCD3 or PEX14 from (A) with at least 30 cells imaged per condition. All cells imaged in ABCD3 KD (96% KD), including wildtype cells, were used in quantification. Statistical analysis used unpaired two-tailed t-tests, **** indicates p<0.0001. (D) U2-OS cells stably expressing GFP-ATF6α were treated either with vehicle (DMSO), Tg (100 nM), Tm (2 μg/ml), or Ceapin (6 μM) for 2 hr or 4 hr (shown) prior to fixation, co-staining with anti-ABCD3 and anti-PEX14, and fluorescent imaging. Stress attenuated GFP-ATF6α foci are indicated by arrowheads. Scale bar, 10 μm. (E) Quantification of correlation of GFP-ATF6α and ABCD3 within PEX14 sites.