Figure 6.

SPCA2 activates downstream Wnt/Ca²⁺ signaling. (A) Schematic of downstream activation of Wnt/Ca²⁺ signaling by SPCA2. Store-independent Ca²⁺ entry activated by SPCA2 increases cytoplasmic Ca²⁺, activating CAMKII and inactivating-catenin by phosphorylation, turning Wnt pathway OFF. (B) Representative Western blot of p-CAMKII and p-β-catenin in MDA-MD-231 transfected with vector control or SPCA2R; GAPDH was used as a loading control. (C,D) Quantitation of Western blots (n = 3) by densitometry. (E–G) Ectopic expression of SPCA2 (SPCA2R) in MDA-MB-231 decreased the mesenchymal gene markers, N-cadherin, vimentin, and Zeb1, which were significantly reversed by addition of CAM Kinase II inhibitor (KN 93 phosphate, 20 µM, 24 h). n = 3. (H) Representative live cell Ca²⁺ imaging (2.5× magnification) in calcium-free conditions using Fura2-AM in MDA-MB-231 treated with vehicle (control) or vorinostat (2.5 µM for 24 h). (I) Fluorescence emission ratio of excitation at 340/380 nm showing increase in average resting Ca²⁺ in the presence of vorinostat (n = 28 cells) compared to vehicle control (n = 33 cells). (J) Representative Western blot of non-phosphorylated (active) and total β-catenin in MDA-MD-231 control (scrambled shRNA) and SPCA2 KD, treated with vorinostat as indicated; GAPDH was used as a loading control. (K,L) Quantitation of Western blots by densitometry (n = 3). SPCA2 KD significantly reversed the decrease in active and total levels of β-catenin by vorinostat. Student t-test, *** p < 0.001, ** p < 0.01, * p < 0.05.