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. 2020 Jun 29;10:10555. doi: 10.1038/s41598-020-67550-0

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© The Author(s) 2020

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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MiR-3135b downregulates GOLPH3 and suppresses the phosphorylation of AKT1 and mTOR in HCT-15 cells. (A) Cropped images from immunoblots using antibodies against GOLPH3 (1:500), p-AKT1 (Ser473, 1:1,000) and p-mTOR (Ser2448, 1:1,000) primary antibodies. Anti-α-tubulin antibodies were used as loading control. Complete developed Western blot membranes are provided in Supplementary data 1. (B) Densitometric analysis of immunodetected bands in panel A. Data represent the mean ± S.D. of three independent experiments, One-way ANOVA was applied to compare control conditions measurements among treatments while post-hoc Tukey’s comparison was used to obtain p values. Images are representative of three independent experiments. (C) Schematic representation of p-miR report constructs containing the 3′-UTR of the GOLPH3 cloned downstream of luciferase gene. miR-3135b seed sequence is indicated in gray box. Point mutations in the miR-3135b binding sites of 3′-UTR of the GOLPH3 gene are denoted in bold. (D) Luciferase assays in HCT-15 cells transfected with miR-3135b and the constructs described in panel C. Error bars represent the standard deviation of triplicate experiments and p values < 0.05 were considered to be statistically significant. NS = non-significant.