Ability of CSR3 and CSR3-Ala to interfere with <i>gfp</i> silencing in <i>gfp</i>-transgenic <i>C</i>. <i>elegans</i> (strain RT476) expressing GFP under an intestine-specific promoter.

<p>(<b>A, B</b>) Upper panel: representative images of <i>C</i>. <i>elegans</i> intestine using bright field (BF) illumination or UV light to observe fluorescence of GFP or the red fluorescent protein dTomato. Lower panel: normalized average intensity of GFP fluorescence in four independent experiments. Error bars indicated S.E. (n = 35–50). In (<b>A</b>), the <i>gfp</i>-transgenic strain RT476 was transformed to express CSR3 or CSR3-Ala under a heat shock—inducible promoter, and two transgenic lines expressing each protein were included in experiments. Sense-mediated silencing of <i>gfp</i> expression was induced in the nematodes by feeding them <i>E</i>. <i>coli</i> expressing <i>gfp</i> mRNA (RNAi), which reduced GFP fluorescence significantly (<i>gfp</i>-transgenic nematodes fed bacteria harboring an insert-less plasmid were used as a control) (Student’s t-test, p < 0.001). At 72 h, expression of CSR3 and CSR3-Ala was induced in the <i>gfp</i>-silenced nematodes by heat shock. At 24 h post-induction, however, no significant difference (Student’s t-test) in GFP fluorescence was detected in nematodes expressing CSR3 or CSR3-Ala as compared with the <i>gfp</i>-silenced strain (RNAi). In (<b>B</b>), similar experiments as above were carried out with the <i>gfp</i>-transgenic strain RT476 transformed to express CSR3 or CSR3-Ala fused with dTomato. No recovery of GFP fluorescence was observed. (<b>C</b>) Immunoblot analysis using a rabbit polyclonal antibody specific to CSR3 detected the dTomato-CSR3 fusion protein in nematodes (B) in which dTomato-CSR3 expression was induced. (<b>D</b>) Simultaneous induction of CSR3 expression and GFP-silencing. CSR3 was not able to delay silencing.