XB-IMG-172272
Xenbase Image ID: 172272
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Fig 4. Celf1-mediated post-transcriptional control of mitotic machinery components facilitates lens fiber cell nuclear degradation.(A, A’) Compared to control lens, phosphorylation of Lamin A/C (pLamin A/C) is reduced in Celf1cKO/lacZKI lens. Dotted-line areas of the fiber cell regions in control (B-D) and Celf1cKO/lacZKI mice (B’-D’) lenses are shown at high-magnification. Arrows in B, C indicate examples of high pLamin A/C expressing nuclei, while no nuclei are observed in D as this area represents a normal nuclear-free zone in the control lens. Asterisks in B’-D’ indicate reduced signals of pLamin A/C in Celf1cKO/lacZKI lens fiber cell nuclei. Note the presence of nuclei in D’ in the centrally located fiber cells due to the nuclear degradation defects in the Celf1cKO/lacZKI lens. (E, E’) Unlike in the control lens where p27Kip1 protein is restricted to cells of the transition zone and cortical fiber cells (arrow), high levels of p27Kip1 protein are detected in the entire fiber cell compartment (asterisks) including the central region in Celf1cKO/lacZKI lens. (F, F’) High-magnification of dotted-line areas in E and F. Asterisks indicate elevated signals of p27Kip1 protein. (G) Quantification of the p27Kip1 immunofluorescence signals from control and Celf1cKO/lacZKI lenses shows significantly increased p27Kip1 protein levels in Celf1cKO/lacZKI lenses. (H) Western blot analysis shows increased levels of p27Kip1 protein in Celf1cKO/lacZKI lens compared to control. (I) Compared to control, p27Kip1 mRNA levels are not significantly altered in Celf1cKO/lacZKI lens. (J, K) RIP and CLIP assays, respectively, identify p27Kip1 mRNA to be highly enriched in Celf1-pulldown in wild-type mouse lens. (L) A potential Celf1 binding GU-rich region in present in the mouse p27Kip1 5’ UTR. (M) Activity of firefly luciferase fused downstream of p27Kip1 5’ UTR is significantly elevated in Celf1-knockdown (Celf1-KD) mouse lens cell line compared to control (firefly luciferase normalized to Renilla luciferase (Rluc)). (N-O’) Compared to control, p21Cip1 mRNA and protein is abnormally elevated in Celf1cKO/lacZKI lens. Asterisk in O’ indicate high expression of p21Cip1 protein in the Celf1cKO/lacZKI lens. Abbr.: e, epithelial cells; f, fiber cells; tz, transition zone; NS, not significant. Scale bar in A’, E’, O’ is 75 μm and in D’ and F’ is 12 μm. Asterisks in G, K, M and N indicate a p-value < 0.0005, 0.005, 0.05 and 0.005, respectively. Image published in: Siddam AD et al. (2018) © 2018 Siddam et al. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View |