Article Title: TIM-1 Promotes Japanese Encephalitis Virus Entry and Infection
Figure Lengend Snippet: Knockdown of TIM-1 expression impairs JEV entry and infection. A549 cells were seeded in the 12-well plate, and they were transfected with TIM-1-specific siRNA of different concentrations or control siRNA at 50 nM using lipofectamine RNAiMAX Reagent. At 48 h post-transfection, cells were infected with JEV NJ2008 (MOI = 1) for 24 h. ( A ) Cell lysates were harvested to detect the level of JEV NS1 protein and cellular TIM-1 expression by Western blot analysis, using GAPDH as an internal control. One representative experiment out of three is shown; ( B ) production of progeny virions were determined by titering the supernatants of indicated cells in BHK-21 cells. Data are presented as mean ± SD from three independent experiments; ( C ) cells were transfected with TIM-1 siRNA or control siRNA for 48 h before being challenged with JEV NJ2008 (MOI of 10). At 24 h post-infection, cells were fixed and stained for E glycoprotein (red) and TIM-1 (green), followed by confocal microscopy analysis. Scale bars, 20 µm. A549 cells were transfected with TIM-1 siRNA or control siRNA for 48 h; ( D ) cells were incubated with JEV NJ2008 (MOI of 5) at 4 °C for 30 min and then washed with PBS three times; ( E ) cells were incubated with JEV NJ2008 with a MOI of 5 at 4 °C for 1 h and washed with PBS three times, then shifted to 37 °C for 15 min to allow JEV entry. Cells were treated with proteinase K (1 mg/mL) to remove non-internalized virions. Total RNA was extracted and used for quantification of JEV RNA, the efficiency of TIM-1 silencing and JEV attachment and entry were detected by qRT-PCR. Data are presented as mean ± SD from three independent experiments using t-test. ** p
Article Snippet: Pools of TIM-1 siRNA (sc-61691) and control siRNA (sc-37007) were purchased from SantaCruz.
Techniques: Expressing, Infection, Transfection, Western Blot, Staining, Confocal Microscopy, Incubation, Quantitative RT-PCR