Fig. 3

DVL2 loss of function reduces in vitro Wnt target genes expression, cell proliferation and cell cycle progression ± HER2 inhibition. A, B RT-qPCR based mRNA expression analyses of Wnt target genes (DVL2, DVL3, CTNNB1, CCND1, POU5F1, VEGFA, MMP7, ERBB2, AKT1) respectively in SKBR3 and BT474 cells stably expressing NTC vs. shDVL2 with or without 1 nM Neratinib (Nert) treatments. Transcript levels were normalized using GAPDH, B2M and GUSB as housekeeping control. All experiments were performed in triplicates and Welch’s independent T-test were performed between NTC vs. shDVL2 or NTC vs. NTC + Nert or NTC + Nert vs. shDVL2 + Nert to investigate the additive effect of HER2 inhibition with DVL2 loss of function (n = 3; *p < 0.05). C, D 2D proliferation assay was performed in SKBR3 and BT474 breast cancer cell lines via in vitro live cell image analyses. SKBR3 and BT474 cells stably expressing NTC vs. shDVL2 were seeded in a 96-well plate and upon confluence were treated with 1 nM Nert or DMSO vehicle. Cells were monitored and live cell percentages were quantified in real time via IncuCyte ZOOM (Essen Biosciences) for 144 and 280 h respectively for SKBR3 and BT474 cells. For each time points Mean ± SEM values were shown and considered statistically significant at *p < 0.05. E, F The effect of HER2 inhibition via ± 1 nM Neratinib (Nert) treatment respectively in SKBR3 and BT474 cells stably expressing NTC vs. shDVL2 was analyzed in a cell cycle assay using a Vybrant cell violet dye (Invitrogen) followed by flow cytometry. The live cells are represented in the SSC vs. FSC plots, which were further distributed among different cell cycle stages such as SubG1, G1, S, and G2/M phases. Experiments were performed in triplicates and mean values were shown in the graphs below. One-way Anova with Tukey’s multiple comparison test were performed where different letters denote statistical significance at p < 0.05