Figure 12.

Loss of UBXN1 leads to para-mitochondrial accumulation of MFN2 interfering with PRKN translocation. (A) wild-type (WT) and UBXN1^-/- HeLa cells (#22 and #71) without ectopic PRKN expression were treated with 25 µM CCCP for 6 h. Whole cell lysates were analyzed by western blotting using anti-PINK1, anti-UBXN1 and anti-GAPDH antibodies. (B) wild-type (WT) and HeLa UBXN1^-/- clone 22 (#22) and clone 71 (#71) cells were transfected with an expression plasmid for mCherry-PRKN or control vector, fixed, stained using rabbit anti-MFN2 antibodies and mouse anti-CYCS antibodies and imaged by confocal microscopy. Shown are representative images from three independent experiments. Scale bar: 20 µm. (C) Percentage of cells with MFN2 blobs in mCherry-PRKN or control transfected wild-type, HeLa UBXN1^-/- clone 22 and clone 71 cells. The bar graph shows data from three independent experiments with 28 to 68 cells per condition. Statistical significance was tested by unbalanced two-way ANOVA followed by Student’s t-test with “fdr” adjustment for multiple testing. (D) HeLa cells and cells of HeLa UBXN1^-/- clone 22 (#22) and clone 71 (#71) were transfected with expression plasmid for mCherry-PRKN and treated for 60 min with 25 µM CCCP, fixed, stained using rabbit anti-MFN2 antibodies and mouse anti-CYCS antibodies and imaged by confocal microscopy. Shown are representative images from three independent experiments. Scale bar: 20 µm. (E) Dot blot to depict correlation between MFN2 blob formation and PRKN translocation to depolarized mitochondria in cells of panel D. Data shown are derived from three independent experiments with 81 to 116 cell per condition. Statistical significance was ascertained using the chi-squared test.