Fig. 6

PDE10A inhibition decreases β-catenin, MAPK and AKT oncogenic signaling. A Left: confocal immunofluorescence microscopy of β-catenin and DAPI nuclear stain in SKOV3 cells pre-incubated with 25 μM Pf-2545920 or DMSO then treated with Wnt-3A conditioned media or L-cell media for additional 5 h; Right: quantitation of mean nuclear β-catenin intensity of experiment shown; Far right: quantitation of mean nuclear β-catenin intensity of SKOV3 cells pre-incubated with Pf-2545920 or DMSO for 24 h then stimulated with Wnt3A or L-cell media. Error bars, SD, (n = 3–7 images) ***p < 0.001 (ordinary one-way ANOVA) (B) Subcellular fractionation and detection of β-catenin in cytoplasmic [C], membrane [M], and nuclear [N] compartments of SKOV3 cells pre-incubated with Pf-2545920 or DMSO for 3 h then stimulated with Wnt-3A or L-cell media for additional 5 h. Loading controls were Na + /K + ATPase for membranous, Lamin A/C for nuclear, and GAPDH for cytoplasmic fractions. C Pf-2545920 and MCI-030 decrease β-catenin transcriptional targets in OV-90 and TOV112D cells. GAPDH was used as the loading control. D PDE10A gene KO decreases β-catenin levels and downstream transcriptional targets in OV-90 and SKOV3 cells. GAPDH was used as the loading control. E MCI-030 decreases activation β-catenin, MAPK, and AKT signaling pathways in SKOV3 cells in a time and concentration-dependent manner. GAPDH was used as the loading control. F Pre-incubation of SKOV3 cells with MCI-030 for 24 h inhibit EGF-induced MAPK and AKT signaling pathways (EGF treatment at 30 ng/mL for 5 min). GAPDH was used as the loading control