Figure 7.

BPLF1 promotes the formation of HTTQ109 aggregates in transfected cells. HeLa cells were co-transfected with HTTQ109-GFP and BPLF1, BPLF1[C61A] or empty vector and expression of the transfected proteins was detected by immunofluorescence after 24 h or 48 h. (A) Representative micrographs illustrating the enhanced formation of HTTQ109-GFP aggregates in cells expressing catalytically active BPLF1. Scale bar: 10 μm (B) Quantification of number of aggregate-positive cells. Mean ± SEM of three independent experiments where a minimum of 176 cells were scored for each condition. Statistical analysis was performed using Student t-test. *P ≤ 0.05, **P ≤ 0.01. (C) The accumulation of toxic HTTQ109-GFP in cells expressing catalytically active BPLF1 induces cell death. The number of cells co-expressing HTTQ109-GFP and either BPLF1, BPLF1[C61A] or FLAG-vector was assessed 24 h or 48 h after transfection in three independent experiments. A selective loss of cells co-expressing HTTQ109-GFP and BPLF1 was observed after culture for 48 h. (D) Filter trap assays were performed with lysates of HeLa cells transfected as in A. A significantly stronger and progressive accumulation of HTTQ109-GFP aggregates was observed in cells expressing catalytically active BPLF1. The same lysates were fractionated by SDS-PAGE and immunoblotted to control for BPLF1 expression and loading. Images from one representative experiment out of three are shown. (E) Quantification of data presented in D; means ± SEM of three independent experiments