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. 2020 Oct 19;11:5267. doi: 10.1038/s41467-020-18942-3

Fig. 1. Light-assisted peroxisomal ROS production.

Fig. 1

a Schematic of how we trigger peroxisomal ROS generation by 559 nm light. b, c The peroxisome redox sensor roGFP2-PTS1 was used to confirm peroxisomal ROS production following 559 nm illumination. b Peroxisomes within the white circular region of a NIH3T3 cell expressing diKillerRed-PTS1 and roGFP2-PTS1 were illuminated with 559 nm light (top panels: before 559 nm light illumination; bottom panels: after 559 nm light illumination). Left: roGFP2-PTS1 emission with 488 nm excitation (green), middle: roGFP2-PTS1 emission with 405 nm excitation (cyan). Right: immediate loss of diKillerRed-PTS1 emission following 559 nm illumination. c Quantified changes in roGFP2-PTS1 emission ratios (405/488 nm) following 559 nm illumination (in NIH3T3 cells expressing diKillerRed-PTS1 and roGFP2-PTS1). A 559 nm illumination increased the mean 405/488 nm emission ratio for roGFP2-PTS1 (n = 8 cells). Error bars displayed on graphs represent the mean + SEM. ***P = 1.72E−05 (one-tailed paired samples t test). d Peroxisomes indicated by the white arrows in a NIH3T3 cell expressing EGFP-Ub, PMP34-TagBFP, and diKillerRed-PTS1 were illuminated with 559 nm light (top panels), leading to the immediate loss of their diKillerRed-PTS1 fluorescence. EGFP-Ub later specifically accumulated on 559 nm illuminated peroxisomes (bottom panels: 40 min after 559 nm illumination, n = 31 cells). e–g A NIH3T3 cell expressing diKillerRed-PTS1, EGFP-p62, and PMP34-TagBFP was illuminated with 559 nm at the white circular region, leading to the immediate loss in diKillerRed-PTS1 fluorescence. g EGFP-p62 later accumulated onto the ROS-stressed peroxisomes (bottom panels: magnified view of the white square region in the top panels. Yellow arrowheads indicate EGFP-p62 on damaged peroxisomes; n = 13 cells). h, i A NIH3T3 cell expressing diKillerRed-PTS1, EGFP-LC3B, and PMP34-TagBFP was illuminated with 559 nm at the white circular region, leading to the local appearance of EGFP-LC3B puncta in i. j Magnified view of the white square region in i. EGFP-LC3B specifically accumulated on 559 nm illuminated peroxisomes (white arrows, n = 33 cells). All scale bars, 5 µm. Source data are provided as a Source data file.