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. 2019 Apr 1;9:5446. doi: 10.1038/s41598-019-41940-5

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© The Author(s) 2019

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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Increased canonical Wnt signalling following loss of TMEM67 is dependent on HOXB5. (a) Relative transcript expression levels of TMEM67 (left panel), IFT88 (middle panel) and HOXB5 (right panel), following knockdown of either TMEM67 (siTMEM67) or IFT88 (siIFT88) compared to scrambled negative control (siScr). Statistical significance of pairwise comparisons are indicated by: n.s. not significant; **p < 0.01; for Student’s t-test (paired, two-tailed). Error bars = s.e.m. Values are the mean of n = 3 independent experiments, with qRT-PCR runs performed in duplicate. (b) Increased HOXB5 protein expression following siRNA knock-down of TMEM67 but not IFT88. Relative protein level ratios for HOXB5 and β-actin (loading control) are indicated. (c) HOXB5 nuclear expression (green) is increased following TMEM67 knockdown (green arrowheads). Scale bar = 20 μm. (d) Exogenous over-expression of FLAG-tagged HOXB5 (predicted size 33 kDa), visualized as a double band by immunoblotting (IB) with anti-FLAG (upper panel), increased expression of active β-catenin and total β-catenin. Treatment with Wnt3a conditioned media further increased active β-catenin and total β-catenin levels, compared to L cell control. Relative protein level ratios for HOXB5 and β-actin (loading control) are indicated. Full scans of blots are shown in the Supplementary Information File. (e) TOPFlash reporter assays showing increased canonical Wnt/β-catenin signalling following FLAG-HOXB5 over-expression and treatment with Wnt3a conditioned media, as indicated. (f) Activation of Wnt target genes AXIN2 and DKK1 was dependent on loss of TMEM67 and over-expression of HOXB5. For all panels, statistical significance of pairwise comparisons are indicated by: n.s. not significant; *p < 0.05; **p < 0.01; for Student’s t-test (paired, two-tailed). Error bars = s.e.m. Values are the mean of n = 3 independent experiments.