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. 2013 Aug 19;23(1):145–156. doi: 10.1093/hmg/ddt407

Figure 3.

Figure 3.

Kinase active HK2 promotes depolarization-induced parkin recruitment in HeLa cells and primary cortical neurons. (A) HeLa lines stably expressing YFP-parkin, MitoDsRed2 and either control (left panels) or HK2 (right panels) shRNA transfected with V5-tagged LacZ (upper panels), HK2 WT (middle panels) or HK2 KD (lower panels) constructs and treated with 2 μm CCCP (2 h, low-glucose media). Scale bar: 20 μm. (B) Quantitation from (A) of the mean overlap area between YFP-parkin and MitoDsRed2 in cells co-expressing V5-tagged constructs (n = 6 wells per condition, error bars are SEM). Data from CCCP-treated conditions were analyzed by two-way ANOVA with Bonferroni's post hoc test; ***P < 0.001, versus CCCP-treated control shRNA for each construct. (C) Immunoblots of HK1 and HK2 (filled arrows) in the human frontal cortex, human midbrain and HeLa lysates with cyclophilin B (open arrows) as a loading control. (D) Effects of knockdown of both HK1 and HK2 on parkin recruitment. HeLa cells stably expressing YFP-parkin were transfected with siRNA against PINK1, HK1, HK2 or both HK1 and HK2 and were treated with 2 μm CCCP (2 h, low-glucose media) for 2 h, fixed and analyzed for the mean area of discreet YFP-parkin ‘spots’ per cell (n = 5 wells per condition, error bars are SEM). Data from CCCP-treated conditions were analyzed by one-way ANOVA with Dunnett's post hoc test; ***P < 0.001, significantly different from non-targeting control siRNA condition. (E) HeLa lines stably expressing YFP-parkin, MitoDsRed2 and either control, HK2 or PINK1 shRNA were transfected with V5-tagged LacZ, HK1 WT, HK1 KD, HK2 WT or HK2 KD constructs and treated with 2 μm CCCP (2 h, low-glucose media). Data graphed are the mean overlap area between YFP-parkin and MitoDsRed2 in cells co-expressing V5-tagged constructs (n = 3 wells per condition, error bars are SEM). Data from CCCP-treated conditions were analyzed by two-way ANOVA with Bonferroni's post hoc test; *P < 0.05, **P < 0.01, ***P < 0.001, versus CCCP-treated control shRNA for each construct. (F) Primary cortical neurons transfected with YFP-parkin and V5-tagged LacZ, HK2 WT or HK2 KD constructs were treated with 10 μm CCCP for 6 h, fixed and labeled for TOM20 and V5. Scale bar: 20 μm. (G) Quantification of the proportion of neurons with discreet areas of mitochondria-localized YFP-parkin assessed under blinded conditions. P-values are by chi-squared test.