FIG 2.

ESCRT function is inhibited in inducible ESCRT-III fibroblasts. (A) GFP expression of inducible fibroblasts expressing GFP, CHMP6-GFP, CHMP4B-GFP, or CHMP4C-GFP 24 h post-Dox addition. Nuclei were labeled with DAPI (blue). Bars, 10 μm. (B) Western blot analysis of HCMV-infected cells expressing CHMP6-GFP from the HCMV genome (HCMV) or of fibroblasts following lentiviral transduction (Lenti) and selection for cells containing the inducible construct. Dox was present throughout the infection (96 h). (C) Western blot analysis of endogenous and GFP-tagged CHMP6, CHMP4B, and CHMP4C at 96 hpi after Dox induction for 0, 48, 72, or 96 h. NS, nonspecific band detected by the CHMP4B antibody; *, unknown band detected by the CHMP6 antibody only in Dox-induced samples. (D) EGF degradation in ESCRT-III dominant negative (green) fibroblasts. EGF (red) was imaged 3 h after a 30-min pulse in the presence or absence of Dox. Nuclei are labeled with DAPI. Bars, 10 μm. (E) HSV-1 infection of fibroblasts expressing GFP, CHMP6-GFP, or CHMP4-GFP at 24 hpi (MOI, 5). Dox was added 24 h prior to infection. Significant differences (P < 0.05) between samples are marked with asterisks. Infections were carried out in three independent experiments.