Fig. 6: Organelle and protein alterations in the liver of Acbd5−/− mice.
a Representative EM images of hepatocytes from Acbd5+/+ (WT) and Acbd5−/− mice (n = 3, age 3 months) with peroxisomes labeled by alkaline DAB staining. b Average peroxisome abundance in hepatocytes. c Immunoblots from liver homogenates (n = 5/group) detecting proteins related to pathways involved in cellular lipid metabolism. L-BP—peroxisomal L-bifunctional protein, Thiolase—peroxisomal 3-ketoacyl-CoA thiolase, ATPA—ATP synthase subunit alpha (full size immunoblots are shown in Supplementary Fig. 8). d Relative average intensities of the proteins detected in (c) after normalization against actin. e Peroxisomal protein abundance in subcellular liver fractions (full size immunoblots are shown in Supplementary Fig. 9). PNS—post-nuclear supernatant, HM—heavy mitochondrial fraction, LM—light mitochondrial fraction, FL—fluffy LM layer, MIC—microsomal fraction, CYT—cytosol, G—gradient fractions (density increasing with numbers), L-BP—peroxisomal L-bifunctional protein, ATPA—ATP-synthase, subunit α. Columns G1 (low density)—G5 (high density) represent fractions from the final density gradient. Note that G5 and G4 comprise highly pure peroxisome fractions (see Islinger et al. 2). f Acyl-CoA oxidase activities in PNS and isolated peroxisome fractions (G4 from e). g Total protein yields in peroxisome fraction (G4 + G5) isolated from livers of Acbd5+/+ and Acbd5−/− mice (average from five isolations). h Representative 3D reconstruction of serial EM sections (resolution in x, y, z-axis: 3.5 × 3.5 × 50 nm), peroxisomes are shown in magenta, mitochondria in green and ER cisternae in gray. For estimation of organelle sizes see (a). i Proportion of peroxisomes showing membrane contact sites with at least one other organelle (0.01 mm2/liver from the EM images were used for quantification). j Average minimum distance observed between ER cisternae and peroxisomes or mitochondria calculated from the 3D-reconstructions (all box plots depict the median, interquartile range, and minima/maxima; all bar graphs show data means with standard deviations, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; unpaired t-test, two-sided).
