Figure 6

Cell growth and invasion are increased by ZnR/GPR39. Cell growth, as measured using SRB levels, in cells treated daily with Zn²⁺ (200 μM, 2 min) to activate ZnR/GPR39 versus EDTA (100 μM, 2 min) used to chelate residual or released Zn²⁺. (A) Averaged count of numbers of MCF-7 cells following Zn²⁺ or EDTA daily treatment (p < 0.05 t-test, between treatments for each day). (B) Left panel shows averaged TAMR cell numbers, as in A. (p < 0.05 t-test, between treatments for each day). Middle panel shows the cell numbers in cells pre-treated with the Gαq inhibitor (YM-254890, 10 μM, 30 min) prior to the daily treatment with Zn²⁺ (200 μM, 2 min. n = 5). Bottom right panel shows representative images of the plate following SRB staining on day 0 (no Zn²⁺ treatment) versus day 7 of treatment. Top right panel shows representative Ca²⁺ responses monitored using Fura-2 fluorescence as in Fig. 1, in TAMR cells that were pre-treated with Zn²⁺ (200 μM, 2 min) 24 hrs. prior the imaging (n = 4 coverslips). (C) Analysis of A-B showing the daily fold increase in cell number, as monitored with SRB, following Zn²⁺ treatment compared to EDTA treatment (p < 0.05 t-test, between daily treatment of MCF-7 and TAMR cells). (D) TAMR cells were infected with shGPR39 lentiviral constructs or shSCR (scambled control), as described. Cell growth analysis using SRB was done as in A, and number of cells treated with Zn²⁺ were normalized compared to cells treated with EDTA (as in C; p < 0.05 t-test, between daily treatment of shSCR and shGPR39 cells). Right panel shows representative Ca²⁺ responses monitored using Fura-2 fluorescence in the shGPR39 and shSCR cells, indicating silencing abolished the Zn²⁺-dependent Ca²⁺ signaling. (n = 2 independent experiments (p < 0.05 t-test, between shSCR and shGPR39 each day). (E) Averaged MDA-MB-453 cell numbers for Zn²⁺ or EDTA treatment on each day, the right panel shows representative images of the plate following SRB staining. (p < 0.05 t-test, between treatments) (F) Averaged BT20 cell numbers for Zn²⁺ or EDTA treatment on each day; the right panel shows representative images of the plate following SRB staining. (p < 0.05 t-test, between treatments) (G) Migration of cells via matrigel was determined, for TAMR cells treated with or without the Gαq inhibitor, YM-254890 (1 μM, 30 min). Cells were treated with Zn²⁺ (200 μM, 2 min) or EDTA (100 μM, 2 min) as control, and cells that migrated through Matrigel were counted (20×, DAPI staining, inversed greyscale image). The average numbers of cells are shown in the bar graph (left panel). Right panel shows a representative image of the cells that invaded through Matrigel at 72 h following treatment (Zn²⁺ or EDTA). (n = 3 independent experiments, p < 0.05 ANOVA). (H) Migration of MCF-7 cells using the paradigm described in F.