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. 2019 Jan 23;40(9):1086–1098. doi: 10.1093/carcin/bgz007

Figure 1.

Figure 1.

BVES loss increases Wnt signaling and β-catenin protein levels. (A) TopFlash activity in 293STF cells following BVES shRNA knockdown. (B) AXIN2 fold change by qPCR in 293STF cells with BVES shRNA knockdown. Cells were treated with 50% L-cell-conditioned media or 50% Wnt3a-conditioned media for 16 h before isolation. (C) Cytoplasmic fractions of 293STF cells following shRNA knockdown of BVES. Cells were treated with 50% L-cell-conditioned media or 50% Wnt3a-conditioned media for 2 h before harvest. (D) TopFlash activity in 293STF cells following siRNA knockdown. (E) AXIN2 fold change by qPCR in 293STF cells with siRNA knockdown. Cells were treated with 50% L-cell-conditioned media or 50% Wnt3a-conditioned media for 16 h before isolation. (F) Cytoplasmic fractions of 293STF cells following siRNA knockdown of BVES. Cells were treated with 50% Wnt3a-conditioned media for 2 h before harvest. (G) β-catenin (CTNNB1) transcript levels in 293STF cells 56–72 h following BVES siRNA knockdown. (H) 293STF cells were treated with 20 μM MG132 for 4 h followed by cytoplasmic fractionation. Black arrow indicates β-catenin, and the relative intensity of only this lower band is quantified below. Bracket indicates ubiquitinated species. Western blot is representative of two independent experiments. (I) 293STF cells were treated with 100 μg/ml cycloheximide (CHX) dissolved in dimethyl sulfoxide (DMSO) or DMSO vehicle control for the indicated time points followed by cytoplasmic fractionation. Western blot is representative of two independent experiments. For (A), (B), (D) and (E), data are representative of at least four independent experiments. **P < 0.01, ****P < 0.0001 by Student’s t-test in the minus Wnt and plus Wnt conditions. For (C) and (F), data are pooled from n = 5 independent experiments, each normalized to the control minus Wnt3a condition. **P < 0.01 by Mann–Whitney test in the minus Wnt and plus Wnt conditions. For (G), data are pooled from n = 4 independent experiments. ns = non-significant by Mann–Whitney test in the minus Wnt and plus Wnt conditions.