FANCL-suppression reduces β-catenin expression and activity. (A) pLK0.1 FANCL shRNA constructs and a control shRNA construct (scrambled, scr) were tested for their ability to knockdown exogenous (top panel) and endogenous (bottom panel) expression of FANCL in 293FT cells, by Western blot and qRT-PCR analysis (mean ± SEM), respectively. FANCL shRNA constructs labeled A, B, and C were used for subsequent experiments. (B) Importantly, suppression of FANCL expression reproduces the classic FA phenotype of excessive radial formation (filled arrow head) and chromosome breaks (unfilled arrow head) with mitomycin C treatment compared with scrambled control. The table summarizes the average from 2 separate experiments. (C) Nuclear extracts from cells expressing vector-control (VC) or scrambled shRNA or FANCL shRNA (construct B) were immunoblotted for β-catenin. Shown is a representative blot from 7 experiments (blot). We performed qRT-PCR to determine whether these changes are associated with changes in β-catenin mRNA. Shown are the combined results of FANCL shRNA construct A, B, and C (graph, mean ± SEM). (D) FANCL was suppressed by shRNA expression in 293FT cells stably expressing scramble-eGFP (closed markers) or LEF-TCF-eGFP (open markers) reporters. The cells were assayed for their ability to respond to BIO stimulation using eGFP as the readout. Relative mean fluorescence intensity (MFI) was calculated relative to the no BIO condition. Shown is the average from 2 experiments with multiple independent replicates in each experiment. (E) qRT-PCR analysis of Wnt-responsive targets in the same cells generated from the experiments described in panel D. Horizontal bars indicate mean ± SEM. See “Methods” for details on qRT-PCR data analysis.