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. 2016 May 1;8:132–149. doi: 10.1016/j.ebiom.2016.04.037

Fig. 6.

Fig. 6

Accumulated β-catenin in BRAFi resistant melanoma cell lines acts independent of the canonical Wnt signaling pathway and the TCF/LEF factors.

a) TOPflash luciferase reporter assays were done to measure the transcriptional activity of TCF/LEF complexes. Sensitive (black bars) and resistant (red bars) melanoma cell lines were transfected with the reporter construct plus CMV-renilla luciferase as a normalization control and treated for 24 h with the indicated concentrations of vemurafenib. For the induction of the full signaling activity a pre-treatment with 15 mM LiCl was performed. Firefly luciferase activity was normalized to renilla activity. Mean values and standard deviations of six samples are presented. Multiple t-tests with Holm–Šídák correction were used to compare data of sensitive and resistant samples and p < 0.05 was considered as significant (asterisk).

b) Co-immunoprecipitation experiments for the detection of interactions of β-catenin with TCF4, LEF1 and Mitf. Soluble lysates were prepared from the indicated sensitive and resistant melanoma cells and incubated with an immobilized β-catenin specific nanobody. Input, non-bound and bound fractions were separated on a SDS-PAGE followed by immunoblot analysis for the transcription factors TCF4, LEF1 and MITF.

c) Cell viability assay (MUH) for testing the effects of released β-catenin from the complexes with TCF/LEF by 50 nM PKF115–584 (circles with dashed curve). The inhibitor was pre-incubated for 6 h before the treatment with increasing concentrations of vemurafenib for 72 h. The assay was measured in quintuplicates. Mean values +/− SD are shown. Multiple t-tests with Holm–Šídák correction were used to compare data points of the two curves and p < 0.05 was considered as significant (asterisk).

d) TOPFlash assay was used to investigate the influence of vemurafenib on β-catenin /TCF/LEF dependent transcription. Reporter transfected cells were treated for 24 h with the indicated concentrations of vemurafenib before assaying the luciferase activity. Firefly values were normalized to renilla signals and to the corresponding untreated controls. Mean values and standard deviations of sixtuplicates are presented. Multiple t-tests with Holm–Šídák correction were used to compare data of sensitive and resistant samples and p < 0.05 was considered as significant (asterisk).