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. 2021 May 10;4:540. doi: 10.1038/s42003-021-02071-8

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — a RNA immunoprecipitation (RIP) assay showing the binding of exogenous FLAG-tagged FMRP and STAT3 mRNA in HCCLM3 cells. b The interaction of endogenous FMRP with STAT3 mRNA in HCCLM3 cells assessed by RIP-qPCR. Error bars represent ±s.d. ***P < 0.001 (IgG compared with anti-FMRP) by two-tailed Student’s t-test. c The interaction of endogenous FMRP with STAT3 mRNA in the protrusions of HCCLM3 cells assessed by RIP-qPCR. Error bars represent ± s.d. ***P < 0.001 (IgG compared with anti-FMRP) by two-tailed Student’s t-test. d FISH assay displaying the colocalization of FMRP and STAT3 mRNA in the cytoplasm and protrusions of HCCLM3 cells. Scale bar: 50 μm. e Quantitative analysis of colocalization of d. The colocalization image was converted into a visual scatter plot. Most of the points are distributed on the diagonal, and the Pearson coefficient is 0.7374. In this case, it indicated that FMRP and STAT3 mRNA are colocalized. f FMRP binds to the 3′UTR of STAT3 mRNA. Aliquots of ³²P-labeled 3′ UTR of STAT3 mRNA were incubated with recombinant FMRP. RNA-protein complexes (indicated by arrow) were formed when the RNA probe incubated with recombinant FMRP. The complexes were competed by 500× excess of unlabeled 3′ UTR of STAT3 mRNA, but not by the non-specific RNA (random tRNA). g The complexes of ³²P-labeled 3′ UTR of STAT3 mRNA with FMRP were competed by 500× excess of unlabeled STAT3 3′ UTR MUT-2, but not by the unlabeled STAT3 3′ UTR MUT-1 and non-specific RNA (Random tRNA). h, i Stable knockdown of FMRP in HCCLM3 cells by lentiviral shRNA sequences (shFMRP). The knockdown effect was verified at both the mRNA and protein levels. j FISH imaging of STAT3 mRNA showing that knockdown of FMRP markedly abolished the protrusion-localization of STAT3 mRNA. The right panel is the quantitative analysis. Number of cells with protrusion signals was reported as a percentage of total cells. Scale bar: 50 μm. The values in the graphs represent the mean of three biologically independent experiments. Error bars represent ±s.d. *P < 0.05, **P < 0.01, ***P < 0.001 by two-tailed Student’s t-test.