Figure 8.

NPEPPS-GFP colocalizes with Etf-1 in cotransfected cells, traffics to E. chaffeensis inclusions, and reduces aggregation of Q103-HTT. (A and B) NPEPPS/PSA-GFP colocalizes with Etf-1 in cotransfected cells and surrounds E. chaffeensis inclusions. (A) DH82 cells were sequentially transfected first with Etf-1 and 1 d later with NPEPPS-GFP. At 1 d p.t. with NPEPPS-GFP, cells were immunostained with anti-Etf-1 (AF555). White arrows indicate the colocalization between the 2 proteins. (B) E. chaffeensis-infected RF/6A cells were transfected with NPEPPS-GFP at 1 d p.i. and stained with DAPI at 1 d p.t. (2 d p.i.). Merged/DIC, fluorescence image merged with DIC image. The boxed area is enlarged on the right. Scale bars: 15 μm. (C) PAQ-22 inhibits E. chaffeensis replication in THP-1 cells. E. chaffeensis-infected THP-1 cells were incubated with 0.1% DMSO (control) or 10 or 100 μM PAQ-22. qPCR of E. chaffeensis 16S rDNA normalized to human GAPDH. *, Significantly different by the Tukey HSD test (P< 0.05). (D to F) Etf-1 reduces aggregation of Q103-GFP. (D) RF/6A cells transfected with Q103-GFP alone or cotransfected with Q103-GFP and DsRed control; exposure time, 0.001 sec. RF/6A cells cotransfected with Q103-GFP and Etf-1 were immunostained with anti-Etf-1 (red); exposure time, 0.6 sec. Scale bars: 15 μm. (E) Percentage of RF/6A cells with Q103-GFP aggregation in cells cotransfected with Q103-GFP and vector control or Etf-1. *, Significantly different by the Tukey HSD test (P < 0.05). (F) Relative amount of Q103-GFP in RF/6A cells with or without Etf-1. Western blot analysis was performed using anti-GFP and - ACT/actin IgG, and band intensities were normalized against ACT/actin.