Figure 5.

Figure 5.

ROME protein negatively regulates the Wnt pathway and calcium signaling in human cancer cells. A and B, Compared with EV control cells, TC-71 (A) and TC-32 (B) cells stably overexpressing ROME were less responsive to recombinant human Wnt3a protein as measured by TOPFlash β-catenin–responsive luciferase reporter. C, TC-71 cells with ROME knockdown by siRNA were more responsive to Wnt3a. D, Endogenous Wnt pathway activity in the colorectal cancer cell line HCT116 was significantly decreased by ROME expression (Western blots confirming ROME-HA expression on the right). Statistical significance was calculated by an unpaired t test (two-tailed) for A–D. E, ROME co-immunoprecipitates with β-catenin in β-catenin pulldown experiments. F, β-Catenin co-immunoprecipitates with ROME in ROME pulldown experiments. G, SPR sensorgrams showing direct binding between the recombinant full-length ROME protein and β-catenin protein. β-Catenin was immobilized on the surface and full-length ROME protein was injected in duplicate at concentrations of 1,250, 416.7, 138.9, 46.3, 15.4, and 5.12 nmol/L. The red lines are the actual data, and the black lines indicate the curve fit. H, ROME expression decreases β-catenin and TCF4 interaction (measured by co-IP). I–K, The Ca²⁺ response was decreased in A4573, STA-ET-7.2, and TC-71 cells overexpressing ROME compared with that in EV-transfected cells when the cells were stimulated with ATP or FBS after overnight serum starvation. L, The Ca²⁺ response was greater in the TC-71 cells with ROME KO than in the WT cells. Statistical significance was calculated by an unpaired t test (two-tailed) for I–L.