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. 2014 Apr 28;205(2):163–177. doi: 10.1083/jcb.201307116

Figure 2.

Figure 2.

Effects of LIC RNAi on Ad5 transport. (A) Protein levels in lysates of A549 cells transfected with control, LIC1-, or LIC2-targeted siRNAs for 48 h evaluated by immunoblotting using anti-p150Glued, anti-IC, and anti–pan-LIC antibodies, with anti-actin as a loading control. LIC1 was reduced by 98%, and LIC2 was reduced by 87%. (Note that moderate increase of LIC2 levels in LIC1 knockdown lane, and vice versa, suggests possible compensatory up-regulation.) (B) LIC RNAi effect on adenovirus infectivity. Ad5-GFP infectivity was examined in A549 cells transfected with LIC1, LIC2, or control siRNAs 48 h before infection with antibody-labeled Ad5 (see Materials and methods). At 24 h p.i., fixed cells were counterstained for DNA (DAPI). LIC1 siRNA inhibited Ad5 infectivity, an effect not detected for control or LIC2 siRNA. Bar, 200 µm. (C) Quantification of virus infectivity shown in B. Mean GFP fluorescence per field of view at 24 h p.i. ± SD from three independent experiments. (D) LIC RNAi effect on adenovirus redistribution. A549 cells expressing LIC1 or control shRNA for 48 h were infected with Ad5 and fixed at 60 min p.i. Cells were immunostained for Ad5 and GFP and counterstained for DNA (DAPI). Adenovirus normally redistributes to the nucleus (control) but remains in the cell periphery in LIC1 RNAi cells. Lines define cell outlines. Bar, 20 µm. (E) Quantification of virus redistribution shown in D. Mean percentage of Ad5 particles localizing at the nucleus 60 min p.i. ± SD from three independent experiments with >30 cells each. LIC1 shRNA inhibited Ad5 redistribution similar to 10 µM nocodazole treatment, an effect not detected for control or LIC2 shRNA. (F) LIC1 RNAi effect on virus run length. A549 cells treated with LIC1 or control siRNA or mock treated were infected with Alexa Fluor 546–labeled Ad5 48 h later and imaged for particle tracking (see Materials and methods). LIC1 siRNA showed a significant reduction in virus run length. Error bars show SDs. **, P < 0.01. A.U., arbitrary unit; ctr, control; noc, nocodazole.