Fig. 1

LONP2 knockdown triggered the accumulation of its substrate TYSND1, alterations in peroxisome size and numbers, and luminal protein import failure. a–d Immunoblotting (a, c) and representative confocal images and quantification of peroxisome size and numbers (b, d) in non-targeting (NT)- and LONP2-silenced COS-7 cells (a, b) and U2OS cells (c, d) after 144 h. TYSND1-F and TYSND1-C indicate full-length TYSND1 and the self-cleaved C-terminal region of TYSND1, respectively. Scale bar, 5 µm. e, f Representative confocal images (e) and quantification (f) of the colocalization between peroxisomes (PMP70) and transiently expressed peroxisome luminal protein (CFP-SKL) in NT- and LONP2-silenced COS-7 cells after 144 h. Scale bar, 2 µm. g Representative confocal images showing the colocalization between peroxisomes (PMP70) and transiently expressed peroxisome membrane protein (PEX3-YFP) in NT- and LONP2-silenced COS-7 cells after 144 h. Scale bar, 5 µm. h Immunoblotting for autophagy-related proteins, vinculin was used as a loading control. i Representative confocal images of COS-7 cells grown in HBSS for 24 h (left) and quantification (right) showing the number of LC3-P62 puncta on peroxisomes of NT- and LONP2-silenced COS-7 cells and U2OS cells after 144 h. Cells were grown in either DMEM or HBSS for 24 h and LC3-P62 puncta on peroxisome was visualized with transiently expressed GFP-LC3 and the immunofluorescence of p62 and PMP70. Scale bar, 2 µm. j Representative confocal images and quantification showing the size and mean branch length of mitochondria in NT- and LONP2-silenced COS-7 cells after 144 h. Scale bar, 5 µm. On all plots, dots represent individual cells from n = 3 technically independent experiments depicted in different colours, and triangles represent the mean within an independent experiment. The mean values were used to calculate the average (horizontal bar), s.d. (error bars), and P-values (using a two-tailed unpaired Student’s t-test)