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. 2008 Nov 27;18(4):737–752. doi: 10.1093/hmg/ddn404

Figure 3.

Figure 3.

Mitochondrial fragmentation and ATP levels of HeLa cells transiently transfected with Htt constructs containing different lengths of polyglutamine repeats. HeLa cells were transfected with plasmid constructs encoding either GFP-, FLAG-tagged or untagged Htt expression constructs containing different lengths of polyglutamine repeats. After transfection, the cells were grown for 18 h in 1.25% FBS and then fixed and immunostained for Tom20 and for the FLAG-expressed protein, where appropriate. Mitochondrial fragmentation in these experiments was quantified by counting between 550 and 1050 cells that stained positive for expression of either the tagged (GFP or FLAG) or untagged Htt-expressed proteins. (A) Representative microscopy images of GFP fluorescence (left panels) and Tom20 staining (right panels) of cells transfected with GFP-Htt28Q (upper panels) or GFP-Htt74Q (lower panels) expression plasmids. Note that the cells expressing GFP-Htt28Q have many tubular mitochondria, whereas those expressing GFP-Htt74Q have numerous small fragmented mitochondria. Bar: 10 µm. (B) Quantification of GFP-expressing cells containing fragmented mitochondria (% of total) following transfection with GFP-Htt28Q and GFP-Htt74Q expression constructs and growth in 1.25% FBS. Data are shown as mean ± SDM (*P < 0.001). (C) Quantification of FLAG-expressing cells containing fragmented mitochondria (% of total) following transfection with FLAG-Htt17Q and FLAG-Htt138Q expression constructs and growth in 1.25% FBS. Data are shown as mean ± SDM (*P < 0.005). (D) HeLa cells were either mock transfected (control) or transfected with untagged Htt17Q or Htt138Q expression constructs. After 24 h, lysates were prepared from the transfected cells and equal amounts of protein were immunoblotted with an anti-huntingtin (upper panel) or anti-actin (lower panel) antibodies. (E) Representative microscopy images of mitochondria visualized by Tom20 staining (right panels) in cells transfected with either Htt17Q (upper panels) or Htt138Q (lower panels) detected with an N-terminal Htt antibody (left panels). Bar: 10 µm. (F) Quantification of the number of transfected cells expressing untagged Htt18Q or Htt138Q proteins cells with fragmented mitochondria grown in conditions similar to (A). Data are shown as mean ± SDM (*P < 0.01). (G) Normalized ATP levels measured in extracts prepared from GFP-Htt28Q or GFP-Htt74Q transfected cells. Data are shown as mean ± SDM (*P < 0.05).