XB-IMG-129964
Xenbase Image ID: 129964
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Fig. 5. Tsc1 is a target of miRNA activity in proximal tubular cells in Xenopus and LLC-PK1 cells. (A) Flowchart of the in silico analysis identifying in vivo relevant miRNA/mRNA target pairs. (B) Table of conserved and mouse kidney-specific miRNAs potentially regulating mTORC1 pathway components. (C) Total number of proximal tubular cells of uninjected controls and MO-injected embryos at stage 42. Number of embryos analyzed and the SD is indicated in the individual bars. *P < 0.005; ns, a not statistical significant change. (D–G) pS6 immunofluorescence comparing the proximal tubules of Dicer, Tsc1, and Tsc1/Dicer double morphants. (H and H′) Tsc1 immunofluorescence of uninjected controls and xDicer-MO–injected embryos. (I) Western blot analysis of LLC-PK1 cells lipofected with sDicer-MO, sTsc1-sMO, and a combination of both MOs using pS6 and α-actin antibodies. (J) Schematic of the Luciferase reporter (pmirGLO-Tsc1) containing the most proximal portion of the Tsc1 3′ UTR. Black bars indicate the predicted miRNA-binding sites. The miR-19a/b (dark blue) and miR-130a/b (light blue) were identified in our in silico analysis and were selected for further analysis because of their dual binding sites. (K) Luciferase assays of LLC-PK1 cells transfected with the empty vector, pmirGLO-Tsc1 alone or in combination with lipofected sDicer-MO, miR-19b, or miR-130b miRNA mimics. SD is indicated in the individual bars. *P < 0.005. (L and L′) Model of mTORC1 signaling, in which the presence (L) or absence of miRNAs (L′) modulates the abundance of Tsc1. Image published in: Romaker D et al. (2014) Copyright © 2014. Image reproduced with permission of the publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License.
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