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. 2017 Jul 6;7:4770. doi: 10.1038/s41598-017-05047-z

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© The Author(s) 2017

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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Wnt signaling activation is inhibited by RA in TKI resistant NSCLC cell line. (a) Left panel, representative Western Blotting analysis of total and active-CTNNB1 in H-1975 treated with 1 and 10 µM RA for 48 and 72 hrs (n = 3). Right panel, densitometric analysis of total and active-CTNNB1 protein levels in H-1975 cells upon RA treatment for 48 and 72 hrs. The analysis has been performed by using GAPDH as normalizer loading control (n = 3) (*=0.02, **=0.05). (b) Immunofluorescence staining of CTNNB1 in H-1975 cells treated with or not with 1 µM RA for 24 hrs (see also insets for all the experimental conditions). Cells are counterstained with Actingreen 488 (green label) and nuclei are marked in blue with DAPI (scale bar 50 µm, n = 3). (c) qRT-PCR analysis of Wnt target genes and the Wnt inhibitor SFRP4 in H-1975 cells treated with 1 and 10 µM RA for 48 hrs. The comparison has been conducted by using the ΔΔCT method and normalized to GAPDH transcript. Dotted line represents the normalized expression levels of each transcript analyzed in untreated cells. Data are represented as mean ± SD. Statistical analysis has been obtained by unpaired t test (n = 3). (d) Luciferase assay of H-1975 cells transfected with Wnt reporters TOPFlash/FOPFlash and treated with 1 and 10 µM RA for 48 hrs. Data are represented as mean ± SD. The histograms represent the value obtained by calculating the Firefly/Renilla ratio, the latter used to measure transfection efficiency. Dotted line represents the normalized expression levels of luciferase activity in H-1975 untreated cells. Statistical analysis has been obtained by unpaired t-test (n = 3). (e) Representative Western Blotting analysis of total and active-CTNNB1 protein levels in H-1975 cells treated with 10 µM RA, 2.5 mM LiCl or co-treated (10 µM RA + 2.5 mM LiCl) for 72 hrs and its relative densitometric analysis (central and right panel). The analysis has been performed by using GAPDH as normalizer loading control (n = 3).