Figure 4

Ubc knockdown affects polyglutamine aggregation in HEK293 cells. Knockdown of ubiquitin conjugating enzymes in HEK293 cells was achieved using short hairpin RNA plasmids (shRNA). HEK293 cells were transfected with Q81:YFP plasmid plus either control or Ubc shRNA plasmids. Two different shRNA constructs were used for each Ubc (designated as 1 and 2 in panels B-D). Cells were allowed to grow for 72 hrs after transfection. A) Following incubation, RNAi silenced cells were imaged by fluorescence microscopy. The pictures shown are an overlay between a bright field and a fluorescent image. B) Western blotting was performed to detect the level of knockdown of each Ubc using the antibodies indicated. All shRNA constructs achieved significant knockdown of their cognate Ubc. C) Aggregate size was measured after Ubc RNAi. Ube2A knockdown leads to smaller Q81:YFP aggregates however, UbcH5b and E2-25K knockdown increase the size of aggregates. At least 150 aggregates were measured for each shRNA transfection. These data represent the average sizes of aggregates from three independent experiments (> 450 aggregates). D) The number of aggregates per cell after Ubc RNAi was determined by analyzing photomicrographs of transfected cells. In control cells, approximately 42% of cells contain only soluble GFP and no aggregates. Ube2A knockdown causes a higher percentage of cells to form aggregates and also results in an increase in the percentage of cells with more than one aggregate. Knockdown of UbcH5b or E2-25K does not alter the ratio of cells containing aggregates versus cells with soluble GFP. Nor do they significantly affect the average number of cells per aggregate. Data were obtained from three independent experiments, counting 150 cells each time.