Figure S1. Expression of
nhr-114 mRNA and Transgenic Intestinal FLAG::NHR-114 (A-C) Northern blot analysis using antisense
nhr-114 probe (described in Figure 1A). (A) In wild-type hermaphrodites, the antisense probe hybridizes to a single product. No signal is present in
nhr-114(
gk849) mutants, whereas two different products are present in
nhr-114(
ef24) mutants (green triangles, kb: kilobases). The smaller one is consistent with a 3'end truncation and the larger one is consistent with a 3'end formation defect, such as an alternative choice of polyadenylation sites. 18S rRNA serves as loading control;
inf-1 mRNA is an internal control for a germline-enriched gene. (B)
nhr-114 mRNA is more abundant in adult hermaphrodites with germ cells than in those without germ cells. Males are
him-8(
e1489), females are
fog-1(253ts). Wild type hermaphrodites (black symbol) with germ lines (+), and without germ lines
glp-1(
q224ts) hermaphrodites (-). RNA from
nhr-114(
gk849) hermaphrodites serves as a negative control for the
nhr-114 probe. (C) Constant expression of
nhr-114 mRNA through development. E=embryo; L1-L4= larval stages; YA=young adult. (D and E) In situ hybridizations using antisense
nhr-114 probe. (D) Wild-type germ lines show
nhr-114 mRNA expression (purple staining) throughout the germ line.
nhr-114(
gk849) germ lines serve as a negative control and show no
nhr-114 mRNA staining. Asterisk indicates the position of the distal end. (E) Section of extruded intestines. From left to right, DIC microscopy image of intact animal focusing on the most anterior part of the intestine. pv: pharyngeal valve (bracket), i: intestine (dotted line). In wild-type
nhr-114 mRNA (dark purple) enriches to the nucleoplasm (arrows). nhr- 114
(gk849) intestines serve as a negative control and show no
nhr-114 mRNA in nuclei. Right images are longer incubation times of different intestines. (F-H) Characterization of intestinal NHR-114 transgenic protein. (F) Immunostaining of extruded intestines of the EV441 strain (n>20 animals). anti-FLAG detects FLAG::NHR-114 in intestinal cells DAPI visualizes nuclei. Anterior (top); posterior (bottom). antianillin (ANI-2) outlines apical membranes. Scale bar 50 um. (G) Nuclear enrichment of FLAG::NHR-114 in intestinal cells. DAPI-positive bacterial remnants outline intestinal lumen. Scale bar 50 um. (H) Anti-FLAG and anti-tubulin immunoblot. FLAG::NHR-114 protein levels remain unchanged upon
nhr-114 RNAi knockdown. Lanes 3 and 4 represent two independent experiments. Effectiveness of RNAi knockdown of endogenous
nhr-114 was confirmed by the appearance of steriles in non-transgenic siblings.