Fig. 3. RNAi of VCP alters the distribution of ubiquitinated proteins and inhibits formation of aggresomes. HeLa cells were subjected to RNAi of VCP for 4 days and/or treated with 10 µM MG132 for 6 hours. (Top) Immunofluorescence confocal micrographs of the indicated experimental groups was conducted with antibodies against either mono- and polyubiquitinated proteins (FK2 antibody) (red) or the Rpn12 subunit of the 26S proteasome (green). (Bottom) Cells for the control and each experimental group were scored for the number of cells with one of four characteristic ubiquitin labelling patterns: (1) no aggregates; (2) dispersed aggregates; (3) single aggresomes; and (4) heavy aggregates. A typical cell for each labelling pattern is indicated by the correspondingly identified arrowhead in the top row of the top panel. The relative abundance of each labelling pattern within a given group of cells is expressed as a percentage of scored cells (n indicates the number of scored cells). The distribution of patterns within each group is significantly different from another with P<0.05 based on a ² test. Scale bar, 20 µm.