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First published online 2 December 2003
doi: 10.1242/jcs.00841

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RNA interference of valosin-containing protein (VCP/p97) reveals multiple cellular roles linked to ubiquitin/proteasome-dependent proteolysis

Cezary Wójcik^1,*, Mihiro Yano^1, and George N. DeMartino^1,

¹ Department of Physiology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9040, USA

View larger version (136K): [in a new window]   Fig. 5. RNAi of VCP induces vacuolization of the cytoplasm. HeLa cells were subjected to RNAi of VCP for 4 days and/or treated with 10 µM MG132 for 6 hours. Immunofluorescence confocal microscopy was conduced for VCP (red) and for p62 (an ER marker protein), ß-COP (a cis-Golgi marker protein), TGN46 (a trans-Golgi marker protein) and Rpn12 (a subunit of the 26S proteasome) (green). Merged images are shown. Single channel images are available online (Figs S1, S2, S3 and S4, http://jcs.biologists.org/supplemental/). Scale bar, 20 µm.

View larger version (53K): [in a new window]   Fig. 1. RNAi of VCP and VCP adaptor proteins in Drosophila S2 cells. Drosophila S2 cells were subjected to RNAi of the indicated proteins. (A) The mRNA levels of indicated RNAi targets were determined by semiquantitative reverse-transcription PCR 4 days after the addition of indicated dsRNAs. (B) Equal amounts of whole-cell lysate protein were subjected to western blotting using antibodies against ubiquitin, dVCP and actin.

View larger version (40K): [in a new window]   Fig. 2. RNAi of VCP, p47, Ufd1 and ß5 subunit of the proteasome in HeLa cells. HeLa cells were subjected to RNAi of the indicated proteins for 4 days or treated with 5 µM MG132 for 24 hours. (A) Equal amounts of whole cell lysate protein were subjected to western blotting with the indicated antibodies. The anti-BIP antibody detects both BIP/Grp78 (lower band) and Grp94 (upper band); (B) Ubiquitin levels were quantified by densitometry of western blots. The ubiquitin level from control lysates was assigned a value of 100% and the levels from experimental lysates are expressed as a percentage of the control. Results represent mean values (±s.e.m.) from three independent experiments. The value for each experimental group was compared to the control by Student's t test. The asterisk indicates P<0.05. (C) Lysates were assayed for the chymotrypsin-like activity of the proteasome by hydrolysis of Suc-LLVY-AMC. Activities from control lysates were assigned a value of 100% and activities from experimental samples are expressed as a percentage of the control. Results represent mean values (±s.e.m.) of three independent experiments. Each experimental group was compared to the control by Student's t test. The asterisk indicates P<0.05.

View larger version (77K): [in a new window]   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.

View larger version (101K): [in a new window]   Fig. 4. RNAi of VCP prevents the accumulation of ubiquitinated proteins at the centrosome. HeLa cells were subjected to RNAi of VCP for 4 days and/or treated with 10 µM MG132 for 6 hours. Immunofluorescence confocal microscopy was conducted with the FK2 antibody detecting mono- and polyubiquitinated proteins (red) and anti-pericentrin antibody detecting the centrosome (green). Scale bar, 20 µM.

View larger version (104K): [in a new window]   Fig. 6. RNAi of VCP alters the distribution of p62 and BIP. HeLa cells were subjected to RNAi of VCP for 4 days and/or treated with 10 µM MG132 for 6 hours. Immunofluorescence confocal microscopy was conduced for BIP (red) and for p62 (green). Scale bar, 20 µm.

View larger version (64K): [in a new window]   Fig. 7. RNAi of VCP causes mitotic abnormalities in HeLa cells. HeLa cells were subjected to RNAi of VCP for 2 days. Compound images of mitotic cells from control and RNAi cells were labelled with anti--tubulin (red) and the DNA dye Yo-Pro iodide (green). Scale bar, 20 µm.

View larger version (26K): [in a new window]   Fig. 8. RNAi of VCP induces apoptosis. (A) HeLa cells were subjected to RNAi of VCP for 2 days or 4 days. Equal amounts of protein from whole cell lysates were subjected to western blotting for the indicated proteins. (B) HeLa cells were subjected to RNAi for the indicated proteins for 4 days or treated with 5 µM MG132 for 6 hours. Caspase 3/7 activity was measured in each group of cells. Activity from control cells was assigned a value of 100% and activities from experimental groups are expressed as a percentage of the control. Results represent the mean (±s.e.m.) of three independent experiments. Each experimental group was compared to the control by Student's t test. The asterisk indicates P<0.05.

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