Figure 5. Up-regulation of AnxA6 in AnxA6 low TNBC cells inhibited ionomycin-induced Ca²⁺ surge and RasGRF2 down-regulation.

(A) MDA-MB-468 cells were transfected with plasmids encoding the empty vector or flag-tagged AnxA6 were grown to 70% confluency, then treated with ionomycin (2 μM) for the indicated time points. Cells were harvested and WCE were used for western blotting and probed with antibodies against AnxA6, RasGRF2, p-ERK1/2 and β-actin (loading control). (B) Densitometric analysis of the effects of ionomycin induced Ca²⁺ surge on the cellular levels of RasGRF2 in control AnxA6 low versus AnxA6 overexpressing TNBC cells from a representative experiment. (C) Determination of the effects of AnxA6 expression on ionomycin induced increase in intracellular Ca²⁺ in Fura-2 loaded control or AnxA6 up-regulated MDA-MB-468 cells by spectrofluorimetry. (D) Effects of ionomycin on the proliferation of control and AnxA6 overexpressing MDA-MB-468 cells. Cell proliferation was determined from quadruplicate samples using the PrestoBlue cell viability assay reagent. ^*indicates p < 0.05.