Figure 3. EB2 promotes Timm23 degradation by cooperating with EB1 in response to CCCP treatment.

(A,B) Only EB2-positive EB1 signals were found to co-localize with fragmented mitochondria. HeLa (Parkin-flag expressed) co-expressing YFP-EB2 (green), DsRed-EB1 (red) and mitochondria-CFP (blue) were treated with 20 μM CCCP or DMSO for 24 h. Triple-positive signals are indicated by arrowheads. Magnified images are shown in the insets. Scale bar, 5 μm. The fluorescence intensities along the dotted arrow were quantified using NIS-Elements AR-Analysis software and presented in panel (B). The length of horizontal dotted arrow in panel B represents distance of dotted arrow in panel (A). (C) Low expression of EB1 and EB2 in EB1 and EB2 knockdown cell lines were confirmed by western blotting respectively. (D–F) Loss of one of EB protein altered the number of foci and percentages of translocation to mitochondria of the other EB proteins in response to CCCP treatment. Cells co-expressing with either CFP-EB1 (green) or CFP-EB2 (green) and mitochondria-DsRed (red) were treated with 20 μM CCCP for 24 h. Scale bar, 5 μm. The number of green and yellow dots per cell and percentages of co-localization with damaged mitochondria (the number of yellow dots/green dots) are shown in panels E and F, respectively. Statistical significance was determined using t tests (n = 19, mean ± SD, *P < 0.05). (G–I) Knocking down of EB2 or EB1 alleviated Timm23 degradation in response to CCCP treatment. The cells were treated with 20 μM CCCP for 24 h and subjected to immunoblot analysis using the indicated antibodies. NC: negative control, EB1 KD: EB1 knockdown cells, EB2 KD: EB2 knockdown cells, Double KD: EB1- and EB2-knockdown cells, EB1 KD + EB1: CFP-EB1-expressing cells with knockdown of endogenous EB1, EB2 KD + EB2: CFP-EB2-expressing cells with knockdown of endogenous EB2.