Figure 5: Effects of VCP-KMT disruption on cellular phenotypes and VCP function.

(a) Representative growth curves of HeLa (left), 293 T-REx Flp-In (middle) and U87-MG cells (right). An exponential fit to the data is shown and the deduced doubling time ô is indicated. (b) Doubling time of wild-type and VCP-KMT-deficient cell lines. Data are represented as means±s.e.m., n is indicated. Significant differences between means are indicated (alpha level 0.05, two-tailed independent two-sample Student's t-test) and P-values are shown. (c) Migration and invasion of wild-type and VCP-KMT-deficient cells. HeLa and U87-MG cells were assayed in a transwell assay with or without Matrigel coating. The percentage of cells having traversed the filter after 24 h is shown. Data are represented as means±s.e.m., n=4. Significant differences between means are indicated (alpha level 0.05, two-tailed independent two-sample Student's t-test) and P-values are shown. (d) VCP content of wild-type and VCP-KMT-deficient cell lines. Whole-cell lysate (7.5 μg protein) was separated by SDS–PAGE and subjected to western blot with VCP- and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-specific antibodies. (e) Localization of VCP in wild-type and VCP-KMT-deficient cells. Fixed and permeabilized cells were stained with VCP-specific antibody. A z-projection of a stack of confocal images is shown. Scale bar=20 μm. (f) Size of endogenous VCP complexes. S100 fractions from HeLa wt and VCP-KMT-deficient cells were separated by SEC and fractions were analysed by anti-VCP western blot. (g) ATPase activity of VCP from wild-type and VCP-KMT-deficient cells. VCP was partially purified from wild-type or VCP-KMT-deficient HeLa cells by anion exchange chromatography and SEC, and the ATPase activity of SEC fractions was measured. (h) Coomassie-stained SDS–PAGE of VCP-containing SEC fractions A–D. Copurified Valyl-tRNA synthetase complex subunits and VCP were identified by MS (arrows).