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. 2014 Nov 1;28(21):2421–2431. doi: 10.1101/gad.241588.114

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© 2014 Foronda et al.; Published by Cold Spring Harbor Laboratory Press

This article, published in Genes & Development, is available under a Creative Commons License (Attribution 4.0 International), as described at http://creativecommons.org/licenses/by/4.0.

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Figure 5. — miR-305 acts via regulation of insulin and Notch pathways in ISCs. (A) Predicted target sites for miR-305 in the 3′ UTRs of the mRNAs encoding InR, PI3K, and Hairless. Residues changed in the target site mutant UTR reporters are shown in red. (B) Effect of miR-305 on luciferase reporters containing the 3′ UTRs of the indicated transcripts. (Ctl) Control luciferase reporter with an SV40 3′ UTR; (UTR) intact 3′ UTR reporters; (Mut) target site-mutated version of the UTR reporter. In all cases, cells were transfected to express miR-305 under the control of the tubulin promoter. Data are the average of four experiments ± SD. P < 0.01 comparing InR, PI3K, or Hairless UTRs with the control UTR; P < 0.025 comparing intact and mutant PI3K UTRs; P < 0.01 for InR and Hairless (Student’s t-test, two-tailed, unequal variance). (C) ISC RNA was TU-tagged by expression of UPRT under Dl-Gal4 in the adult midgut. Dl-Gal4 was combined with Gal80^ts. Animals were reared at 18°C to keep Gal4 inactive and shifted to 29°C at day 5 and aged for 2 d before TU incorporation. InR, PI3K, and Hairless transcript levels were measured by qRT–PCR. Additional replicates are provided in Supplemental Figure S5. (D) esg-Gal4 was used to direct transgene expression in the ISCs and EB cells. esg-Gal4 was combined with Gal80^ts. Animals were reared at 18°C to keep Gal4 inactive and shifted to 29°C at day 2 and aged for 5 d. esg-Gal4-expressing cells (green). (Red) Nuclei labeled with DAPI. (Scatter plot) Ratio of esg-Gal4 cells to total cells. Data were analyzed by ANOVA. (E) Dl-Gal was used to direct transgene expression in the ISCs. Dl-Gal4 was combined with Gal80^ts. Animals were reared at 18°C to keep Gal4 inactive and shifted to 29°C at day 2 and aged for 5 d. The ratio of Dl-Gal4 cells to total cells is shown. Data were analyzed by ANOVA. (F) Dl-Gal was used to direct transgene expression in the ISCs. (Panel 1) Dl-Gal4 + UAS-GFP. (Panel 2) Dl-Gal4 + UAS-GFP + UAS-PI3K. (Panel 3) Dl-Gal4 + UAS-GFP + UAS-Hairless. Dl-Gal4-expressing ISCs (green). Nuclei were labeled with DAPI (red). (Scatter plot) Ratio of Dl-Gal4-expressing to total cells. Data were analyzed by ANOVA.