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. 2006 May 22;173(4):521–532. doi: 10.1083/jcb.200601036

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Copyright © 2006, The Rockefeller University Press

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Figure 7. — Peroxisome biogenesis assay. (A) Schematic representation of the peroxisome photo-chase biogenesis assay. (B) Images of PAGFP-SKL at different time points during the assay. (C) Enlargement of the areas outlined by the stippled boxes in post-1st PA t = 24 h and post-2nd PA in B. Arrows indicate new peroxisomes that were observed only after the second photoactivation event. (D) Distribution of PAGFP-SKL and PEX16-Cerulean after the second photoactivation shows that all of the photoactivated PAGFP-SKL structures contain PEX16-Cerulean. (E) The mean of the total fluorescence intensity (n = 3) attributable to PAGFP-SKL in the PAGFP-SKL/PEX16-Cerulean–expressing NRK cells excited with 488 nm plotted over a 24-h period. Images were collected with a fully open pinhole. The total fluorescence in the cells was calculated by multiplying the mean pixel value of the whole cell with the total number of pixels in the image of the cell. Error bars indicates the standard deviation. n = 3. (F) The mean number of peroxisomes formed by fission versus de novo pathways. The number of new peroxisomes formed by fission was determined by measuring the difference in the number of peroxisomes in the post-1st PA t = 24 h and post-1st PA t = 0 images. The number of peroxisomes formed de novo was determined by measuring the difference between the post-2nd PA and post-1st PA t = 24 h images (see Materials and methods for additional details). The mean number of peroxisomes formed by fission in the 24-h period was calculated as 11 ± 5 (n = 8), whereas a mean of 30 ± 10 (n = 8) new peroxisomes were calculated to be formed de novo. The difference between these two conditions was determined to be statistically significant (P < 0.05; paired t test). Bars, 10 μm.