Fig 1.
Generation and validation of Vcpkmt knockout.
(A) Schematic overview of K315 trimethylation of VCP monomer by VCPKMT. AdoMet = S-adenosyl methionine (B) Targeting of the Vcpkmt gene. Diagram shows the endogenous murine Vcpkmt locus, the position of the targeted gene and the recombined gene. (C) Representative genotyping result from Vcpkmt wild-type (+/+), knockout (-/-) and heterozygous (+/-) mice. (D) Immunostaining for VCPKMT in wild-type and Vcpkmt-/- mouse embryonic fibroblasts. DNA counterstain with DAPI. Scale bars 10 μm. (E) The gene expression of Vcpkmt relative to Gapdh in various murine tissues measured with quantitative RT-PCR. n = 3 mice, means ± SD.
Fig 2.
VCP K315 trimethylation is abolished in Vcpkmt-/- mice tissues.
(A) Mass spectrometric analysis of VCP isolated from wild-type (Vcpkmt+/+) and Vcpkmt-/- mouse tissues. Extracted ion chromatograms corresponding to un-, di- and trimethylated K315 in Arg-C-generated peptide VCP (314–322) are shown. For each tissue the intensity is normalized to the Kme3 signal. Expected elution time of Kme0 (left panel) or Kme3 (right panel) peptides are indicated with an arrow. The data for wild-type tissues have been published previously [6]. (B) Immunoblotting for K315me3-VCP in various wild-type and Vcpkmt-/- tissue protein extracts. 30 μg of whole cell extracts are loaded. Total VCP is shown as a loading control.
Fig 3.
Immunostaining of K315me3-VCP in cells and tissues of wild-type and Vcpkmt-/- mice.
Immunostaining of K315me3-VCP (green), total VCP (red) and DAPI DNA counterstain (blue) of wild-type and Vcpkmt-/-. Scale bars 20 μm. (A) Cultured mouse embryonic fibroblast (MEFs). (B) Semniferous tubuli of testes. (C) Hepatocytes with a central vein. (D) Kidney cortex.
Fig 4.
VCPKMT-mediated in vitro methylation using extracts from Vcpkmt-/- mice tissues and human Vcpkmt-/- cell lines.
Cell extracts were incubated with recombinant VCPKMT in the presence of [3H]AdoMet, proteins separated by SDS-PAGE, and then transferred to a PVDF membrane, which was then subjected to fluorography (FG, lower panels). Extracts were from (A) liver, brain and testis of wild-type and Vcpkmt-/- mice and (B) human wild-type and Vcpkmt-/- cell lines. Western Blot (WB) against VCP serves as loading control (upper panels). w/o = without protein extract.
Fig 5.
Breeding and survival of Vcpkmt-/- mice.
Basic phenotypes of Vcpkmt-/- mice. (A) Sex-ratio of an interbred of Vcpkmt +/- (het) mice. (B) Genotype distribution of het-het breedings compared to an expected Mendelian distribution. +/+ = wild-type, +/- = heterozygous, -/- = knockout. n = 49. Average body weight per genotype of male (C) and female (D) mice with the age of 3, 6, 12 months. Means ±SEM. n = 6–8 mice. (E) Kaplan-Meyer-Plot of the survival of wild-type and Vcpkmt-/- mice monitored over 24 months. n = 25 mice per genotype. (F) Running distance on a treadmill during an acute exhaustion assay. Means ±SEM. n = 6–7 male mice, age 8–10 weeks.