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. 2010 May 14;285(30):23208–23223. doi: 10.1074/jbc.M109.047464

FIGURE 6.

FIGURE 6.

Gene silencing of MAP1LC3β, Beclin 1, or Atg5 by RNA interference inhibits type I interferon signaling by TLR3. Panel A, levels of TLR3-HA, MAP1LC3β, and α-tubulin after MAP1LC3β knockdown are shown. Stable 293 cells expressing WT TLR3-HA were transfected with scrambled control siRNA-A (scrambled siRNA), one of three distinct siRNA duplexes targeting the mRNA of MAP1LC3β (LC3β siRNA-A, -B, or -C), or a version of MAP1LC3β duplex A encoding two silent mutations (Mut. siRNA-A). Equal amounts of protein (40 μg) from the whole cell extracts were then analyzed by SDS-PAGE and immunoblotting with monoclonal anti-HA, anti-α-tubulin, or a polyclonal antibody to MAP1LC3β. The amount of MAP1LC3β in each sample was determined by densitometry using NIH Image 1.63 and expressed relative to the amount of MAP1LC3β in non-transfected (NT) WT cells. Panel B, WT TLR3-HA-expressing cells were co-transfected with pISRE-LUC and either a scrambled control siRNA (control siRNA-A) or one of three MAP1LC3β siRNAs (LC3β siRNA-A, -B, or -C) or mutant LC3β siRNA-A duplex (Mut. siRNA-A) and then stimulated with poly(I:C) (+ I:C, black bars) or vehicle (red bars). The data represent the means ± S.E. of two independent experiments of quadruplicate measurements each. *, p < 0.001 versus all other groups except p = NS versus stimulated WT cells plus control siRNA-A; #, p < 0.001 versus all other groups except stimulated WT; §, p = NS versus other stimulated WT plus an LC3β siRNA except mutant siRNA-A; ¶, p < 0.001 versus all other groups. Panel C, levels of TLR3-HA, Beclin 1, and α-tubulin after Beclin 1 knockdown. WT TLR3-HA cells were transfected with scrambled siRNA-A or one of three siRNAs directed to Beclin 1 (siRNA-A, -B (see supplemental Fig. 5) or -C) or a version of siRNA-C encoding three silent mutations. Equal amounts of protein (30 μg) from the whole cell extracts were then analyzed by SDS-PAGE and immunoblotting with anti-HA, anti-α-tubulin, or a monoclonal antibody to Beclin 1. The amount of Beclin 1 in each sample was determined by densitometry and expressed relative to the amount of Beclin 1 in NT WT TLR3 cells. Panel D, WT TLR3-HA-expressing cells were transfected with pISRE-LUC and either control siRNA-A or one of three siRNA duplexes targeting Beclin 1 (siRNA-A, -B or -C) or mutant siRNA-C and then stimulated with poly(I:C) (+ I:C, black bars) or vehicle (red bars). The data represent the means ± S.E. of two or four independent experiments of eight replicate measurements each. *, p < 0.001 versus unstimulated WT cells or stimulated WT cells plus Beclin 1 siRNAs; p = NS versus stimulated WT plus control siRNA or WT treated with mutant siRNA-C (Mut. siRNA-C); #, p = NS versus stimulated WT; ¶, p < 0.001 versus all other groups except p = NS versus stimulated WT + siRNA-B; §, p < 0.001 versus all other groups except p = NS versus stimulated WT + siRNA-A; ∞, p < 0.001 versus all other groups. Panel E, levels of TLR3-HA, Atg5, and α-tubulin after Atg5 knockdown are shown. Cells expressing WT TLR3 were transfected with scrambled siRNA control or one of two duplexes targeting Atg5 (siRNA-A or -B) or modified versions of the cognate siRNAs encoding four silent mutations (Mut. siRNA-A or -B). Equal amounts of protein (30 μg) were analyzed by SDS-PAGE and immunoblotting with anti-HA, anti-α-tubulin, and monoclonal anti-Atg5 antibodies. The amount of Atg5 in the extracts was determined by densitometry and expressed relative to the amount of Atg5 in non-transfected cells. Note that the migration of Atg5 on SDS-PAGE gels is consistent with its presence in the extracts as the Atg5-Atg12 adduct (∼47 kDa). Panel F, relative luciferase activity in WT TLR3-HA-expressing cells co-transfected with pISRE-LUC reporter and cognate or mutant Atg5 siRNAs is shown. Twenty-four hours post-transfection the cells were stimulated with poly(I:C) (+ I:C, black bars) or vehicle (red bars). The data represent the means ± S.E. of two to four independent experiments of eight replicate measurements each. *, p < 0.001 versus all other groups except stimulated WT cells plus mutant siRNA-B; #, p < 0.001 versus stimulated WT + siRNA-A; p = 0.008 versus stimulated WT plus siRNA-B; p = NS versus stimulated WT + mutant siRNA-A; §, p < 0.001 versus all other groups except p = 0.002 versus stimulated WT cells treated with mutant siRNA-A; ¶, p = 0.002 versus stimulated WT plus siRNA-A; p = NS versus stimulated WT plus control siRNA-A; ∞, p < 0.001 versus stimulated WT + mutant siRNA-B. NT, “non-treated” with siRNA cells (transfected with pISRE-LUC reporter only).