Figure 5
Y260 phosphorylation and α1 Na/K-ATPase/Src interaction in human cancers- (a) Measurement of Y260 phosphorylation in cancer cell lines. Cell lysates from a panel of prostate (LNCAP, DU145, and PC3) and breast (MCF7, MDAMB231, and BT-20) cancer cell lines were compared with corresponding control cells (prostate RWPE1 and breast MCF10A), for Na/K-ATPase α1 expression and Y260 phosphorylation. **p < 0.01compared with respective normal cell line (Unpaired T-test, Welch’s test), n = 3. (b) Interaction between Src and α1 Na/K-ATPase in control DU-P1 and knock-down A4–7 cells analyzed by immunoprecipitation. **p < 0.01 compared with DU-P1(Student’s T-test), n = 4. (c) Effects of α1 Na/K-ATPase knock-down on Src activity, FAK activity and Myc, analyzed by immunoblots. Representative immunoblots are shown and quantitative data from 4–5 separate experiments are presented. *p < 0.05 and **p < 0.01 compared with DU-P1 (Student’s T-test). (d) Measurement of lactate in medium from DU-P1 and A4-7 cell culture (left panel) and in medium from A4-7 cells cultured in the presence or absence of 5 μM PP2 for 4 h (right panel). *p < 0.05 compared with DU-P1 (left panel, n = 4) or A4-7 without PP2 treatment (right panel, n = 3) (Student’s T-test). (e) Cell proliferation rate of DU-P1 vs. A4-7 in 48 hours (left panel) and PP2 inhibition of A4-7 proliferation at 48-hour (right panel). **p < 0.01 compared with untreated control. (f) Effects of α1 Na/K-ATPase knock-down on tumor growth. DU-P1 and A4-7 cells were xenografted into NOD/SCID mice and tumor growth was assessed by measuring the tumor volume at different time points. Photos of tumors harvested from xenografted mice are shown in the upper panel. Quantitative data of tumor weight and volume from xenografted DU-P1 and A4-7 cells are presented in the lower panel. *p < 0.05 and **p < 0.01 compared with basal tumor volume, ##p < 0.01 compared between DU-P1 xenograft and A4-7 xenograft at the same time point (Two-way ANOVA), and **p < 0.01 compared with DU-P1 xenograft weight (Student’s T-test), n = 10.
