Fig. 9. Single EYFP-Stim1 punctae form in similar locations upon multiple rearrangement stimuli. (A) Two EYFP-Stim1-expressing HEK293 cells were imaged by time-lapse TIRFM, during which Ca²⁺ stores were depleted with 2 µM thapsigargin in the presence of nominal extracellular Ca²⁺, 100 µM ML-9 was added to reverse punctae formation and ML-9 was removed to re-stimulate punctae formation. Shown is the fluorescence intensity profile, with each trace representing a single cell. (B) Representative TIRFM images taken at the times indicated (i-iv) in the intensity profile in panel A. The complete image series of this experiment is shown in supplementary material Movie 4. (C) The TIRFM image taken at 380 seconds (store-depleted with thapsigargin before ML-9 treatment) was pseudocolored red. This image was then merged with the image taken at 710 seconds (after ML-9 washout), which was pseudocolored green. Image `b' on the right is a close-up of the region denoted by the white rectangle in the full-size image on the left. Image `a' was generated from the merge of the images 60 seconds before the images used to generate the full-size image on the left, and image `c' was generated from the merge of the images 60 seconds after the images used to generate the full-size image on the left. The arrows in image `b' point to pairs of red and green punctae that remain consistent throughout the series of merged images. Scale bars: 10 µm in B and C; 1 µm in Ca-c.