Journal: Developmental cell

Article Title: Discovery of stromal regulatory networks that suppress Ras-sensitized epithelial cell proliferation

doi: 10.1016/j.devcel.2017.04.024

Figure Lengend Snippet: (A) Differential interference contrast (DIC) images of wild-type C. elegans at mid-larval L4 stage and a schematic diagram of a normal vulva. (bar = 100 μm, high-mag. bar = 10 μm) AC, anchor cell; SG, somatic gonad; M, muscle. Diagram of tissues that are RNAi resistant (rde-1(−)) are indicated in gray; tissues that have been reconstituted with rde-1(+) are indicated in blue. (B) DIC (top) and fluorescence (bottom) images of transgenic worms expressing GFP driven by promoters active in the anchor cell (arrowhead), somatic gonad, muscle, and vulval cells. Dashed lines,worm body boundaries. (C) DIC images of wild-type rde-1 worms (rde-1(+)), rde-1 mutants (rde-1(−)), rde-1(−) mutants with mesoderm-specific RNAi (mesoderm-rde), and rde-1(−) mutants with VPC-specific RNAi (vulva-rde) following lin-3 and lin-39 RNAi. Arrows, normal vulva structures. (D) F1 individuals with tissue-specific expression of let-60(gf) constructs were evaluated for the Muv phenotype. The unc-119(+) allele was used as marker to indicate successful transmission of transgenes. The number of stable transgenic lines generated for each construct is indicated. (E) Offspring for the indicated number of transgenic lines (each bar represents a single independent line; >70 animals were analyzed for each line) were evaluated for the Muv phenotype. Homozygous let-60(n1046) mutants were used for comparison. See also Figure S1 and Table S3.

Article Snippet: C. elegans Tissue-Specific RNAi Strain Construction First-strand cDNA for rde-1 was synthesized from total RNA isolated from N2 wild-type animals, and cDNA for let-60(gf) was synthesized from total RNA isolated from MT2124 let-60(n1046) , with Superscript II Reverse Transcriptase Kit (Invitrogen) following the manufacturer’s protocol. let-60(gf) cDNA was amplified as a single product, and cloned directly into the plasmids (described below).

Techniques: Fluorescence, Transgenic Assay, Expressing, Construct, Marker, Transmission Assay, Generated

Journal: Developmental cell

Article Title: Discovery of stromal regulatory networks that suppress Ras-sensitized epithelial cell proliferation

doi: 10.1016/j.devcel.2017.04.024

Figure Lengend Snippet: (A) Representative DIC and fluorescence images of non-sensitized (gap-1(+)) and sensitized (gap-1(−)) worms expressing a LET-60/RAS-dependent transcriptional reporter. Bottom panels are overexposed to better visualize CFP expression in P5.px, P7.px and P8.px cells. (B) Percentage of gap-1(+) animals with ectopic vulval Ras activity only in presumptive 2° lineage cells following gene-specific or empty vector (control) RNAi. n=44 (control); n>25 (each gene). (C) Percentage of gap-1(−) animals with ectopic vulval Ras activity in both 2° and 3° lineage cells (red bars) or only in presumptive 2° lineage cells (gray bars) following gene-specific or empty vector (control) RNAi. CFP intensity in presumptive 2° lineage cells was scored as high, intermediate (intermed.) and low. n=99 (control); n>25 (each gene). See also Figure S1.

Article Snippet: C. elegans Tissue-Specific RNAi Strain Construction First-strand cDNA for rde-1 was synthesized from total RNA isolated from N2 wild-type animals, and cDNA for let-60(gf) was synthesized from total RNA isolated from MT2124 let-60(n1046) , with Superscript II Reverse Transcriptase Kit (Invitrogen) following the manufacturer’s protocol. let-60(gf) cDNA was amplified as a single product, and cloned directly into the plasmids (described below).

Techniques: Fluorescence, Expressing, Activity Assay, Plasmid Preparation