Figure 4. PP2A reactivation with SMAP reduces AKT-S473 phosphorylation, downregulates RABL6A, and induces PNET cell death in a RABL6A-dependent manner.

(A) Western blots (repeated 3 or more times) of lysates from BON-1 and Qgp1 cells treated 20 hours with SMAP showing effective reduction of pAKT-S473 and pERK-T202/Y204. Loading, vinculin (BON-1), and GAPDH (Qgp1). (B) Representative images from clonogenic assays of BON-1 and Qgp1 cells treated with 0 (vehicle), 5, 10, or 20 μM SMAP for 3 weeks. (C) Quantification of clonogenic assay results, normalized to 100% for vehicle-treated control cells, from 2 separate experiments performed in triplicate. *P < 0.001, 2-way ANOVA and adjusted for multiple comparisons using the Bonferroni method. Identical results were obtained in 4 additional repeats per cell line for cells plated at different densities. (D) Representative experiment (repeated twice) showing dose response curves for BON-1 and Qgp1 cells following exposure for 3 days to indicated concentrations of SMAP. Data represent the mean ± SD for triplicate samples and were normalized to values from untreated cells. (E) Relative cell number (assayed by Cell-Quant) of BON-1 cells expressing CON, KD1, or KD2 shRNAs after exposure for 3 days to increasing concentrations of SMAP. Data (from 3 experiments done in triplicate) were normalized to untreated controls. *P < 0.001 for KD1 and KD2 compared with CON, 2-way ANOVA and adjusted for multiple comparisons using the Bonferroni method. Overall differences between curves were assessed using generalized linear regression. (F) BON-1 CON, KD1, and KD2 cells were treated with 10 μM SMAP for 3 days. Dark and light exposures of Western blots show PP2A activation decreased RABL6A levels and altered its migration on gels. *P < 0.05 for untreated versus treated samples, Student’s t test. Error bars in E and F indicate SEM for data from 3 independent experiments.