1. Uptake of Extracellular Double-Stranded RNA by SID-2
Deborah L. McEwan, Alexandra S. Weisman, Craig P. Hunter 
Molecular Cell 
Volume 47, Issue 5, Pages (September 2012)
DOI: /j.molcel
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2. Molecular Cell 2012 47, 746-754DOI: (10.1016/j.molcel.2012.07.014)
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3. Figure 1
SID-2 Is Required to Internalize Environmental dsRNAs into C. elegans
cy5-labeled dsRNA (500 bp) fed to C. elegans could only be visualized intracellularly in sid-2(+) animals [observed in 6/15 sid-2(+) animals, 0/15 sid-2(–) animals].
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2
SID-2 Expression Is Sufficient for Efficient dsRNA Transport into Drosophila S2 Cells at an Acidic pH
(A) cy5-labeled dsRNA is internalized into and has extensive colocalization with SID-2::GFP expressed in S2 cells in media at pH 5 (42/57 cells contain at least three points of colocalization) but not with cells expressing Cbr-SID-2::GFP (n = 48) or with SID-2::GFP expressed in cells at pH 7 (n = 53). All images were taken at the same time using the same camera settings.
(B) At pH 5, S2 cells expressing SID-2 internalize more 32P-labeled dsRNA than cells expressing Cbr-SID-2 or mock-transfected cells. Results shown are an average of 20 (SID-2, Cbr-SID-2B pH 5), nine (Cbr-SID-2A pH 5), or eight (remaining conditions) biological replicates.
(C) Unlike SID-2, SID-1-dependent transport of 32P-labeled dsRNA is inhibited in media at pH 5 compared to pH 7. Results shown are an average of six biological replicates.
(D) dsRNA internalized by SID-1 or SID-2 is processed into functional siRNAs as indicated by reduced luciferase expression in these cells after the addition of luciferase dsRNA to media at pH 5 (SID-2 and SID-1) or pH 7 (SID-1) as compared to cells not exposed to dsRNA. Results shown are an average of five to eight biological replicates.
∗p < 0.05; ∗∗p < 0.005; ∗∗∗p < , ∗∗∗∗p < 1 × 10−6. Error bars represent 1 standard deviation. p values determined with a two-tailed paired t test. The scale bar represents 10 μm. See also Figure S1 and Table S1.
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5. Figure 3 SID-2-Mediated Transport Is Selective for Long dsRNA
(A) The addition of unlabeled dsRNA competitor blocks the uptake of 32P-labeled dsRNA into cells expressing SID-2, while unlabeled dsDNA has only a moderate effect. Results shown are an average of five biological replicates.
(B–D) Internalization of 32P-labeled substrates into SID-2-expressing, Cbr-SID-2-expressing, or mock-transfected S2 cells. Results shown are an average of nine (100 bp, 25 bp SID-2 and Cbr-SID-2), three (25 bp mock), or six (remaining conditions) biological replicates. Cells expressing Cbr-SID-2 were indistinguishable from mock-transfected cells for all tested substrates.
Error bars represent one standard deviation. Starred conditions accumulated at least 1.4-fold more substrate than mock-transfected cells with ∗p < 0.05; ∗∗p < 0.005; ∗∗∗p < 5 × 10−4. p values determined with a two-tailed paired t test. See also Table S1.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4
Domain Swaps Identify a Critical Role for the SID-2 Extracellular Domain in Environmental RNAi
(A) sid-2(gk505) deletion worms expressing SID-2, Cbr-SID-2, or hybrids of the C. elegans/C. briggsae SID-2 proteins were scored as rescued if exposure to ingested pal-1 dsRNA caused >95% embryonic lethality. Only proteins containing the extracellular SID-2 domain could rescue the environmental RNAi defect.
(B) S2 cells expressing mutagenized SID-2 constructs internalize less 32P-labeled dsRNA than the wild-type (WT) SID-2 control. An exception to this is the triple histidine-to-arginine mutant, which internalizes more 32P-labeled dsRNA than WT. Results shown are an average of five biological replicates. Error bars represent one standard deviation.
See also Figure S2 and Table S1.
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7. Figure 5 Vesicle Trafficking Is Required for SID-2 to Take Up dsRNA
(A) In S2 cells, SID-2-dependent uptake of 32P-labeled dsRNA is a continuous process. Results shown are an average of eight (100 min) or five (remaining conditions) biological replicates.
(B) SID-2-dependent uptake of 32P-labeled dsRNA is strongly reduced by exposing the S2 cells to oligomycin (inhibits ATP synthase), latrunculin A (latA; inhibits actin polymerization), and bafilomycin A1 (bafA; inhibits vesicle maturation); SID-2-expressing cells exposed to these inhibitors internalize less than 2-fold more dsRNA than relevant control cells. Results shown are an average of five (SID-2) or four (Cbr-SID-2) biological replicates.
(C) Endocytosis inhibitors do not prevent SID-1-dependent internalization of 32P-labeled dsRNA; SID-1-expressing cells exposed to these inhibitors internalize between 7.5- and 37-fold more dsRNA than relevant control cells. Results shown are an average of five biological replicates.
(D) LatA inhibits cytoplasmic SID-2::GFP structures and reduces the internalization of cy5-labeled dsRNA (7/50 cells contain at least three points of colocalization between cy5-dsRNA and SID-2::GFP). The scale bar represents 10 μm.
∗p < 0.05; ∗∗p < 0.005; ∗∗∗p < 5 × 10−5. Error bars represent one standard deviation. p values were determined with a two-tailed paired t test.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 6
Model of SID-2 and SID-1 Coordinated Uptake of Ingested dsRNA in C. elegans
dsRNA is internalized from the intestinal lumenal space by SID-2-mediated endocytosis. dsRNA is retained in the vesicle until directly transported into the cytoplasm by SID-1 (left) or released in the pseudocoelomic fluid for subsequent cellular import via SID-1 (right).
Molecular Cell  , DOI: ( /j.molcel )
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