(A,B) rVSMCs were treated with EGF (50 ng/ml) in the presence and absence of EGFR inhibitor gefitinib (Gef, 1 µM) and c-Src inhibitor PP2 (10 µM). (A) TRPM7 expression, normalized to β-actin, after 24 h stimulation and (B) TRPM7 phosphorylation, normalized to β-actin, after 5-min stimulation. Upper panels are representative immunoblots. (C,D) Intracellular Ca²⁺ levels ([Ca²⁺]_i) in rVSMCs induced by EGF (50 ng/ml) were measured in the presence and absence of gefitinib (Gef, 1 µM), the TRPM7 inhibitor NS8593 (NS, 40 µM) and the non-selective cation channel inhibitor 2-APB (30 µM) in rVSMCs. (D) [Ca²⁺]_i data are expressed as (C) fluorescence Cal-520AM-Ca²⁺ signals and (D) calculated as the area under curve. (E) Intracellular free Mg²⁺ ([Mg²⁺]_i ) in rVSMCs after EGF (50 ng/ml) treatment (5 min) in the presence and absence of gefitinib (1 µM), NS8593 (40 µM), apamin (Apa, 1 µM) and 2-APB (30 µM) were measured by Flow Cytometry using specific Mg²⁺ indicator Magnesium Green AM. Raw data were normalized to percentage differences based on the baseline value (veh). (F) ERK1/2 activity was assessed by phosphorylation levels (p-ERK1/2) normalized by total ERK1/2 (t-ERK1/2) expression. Results are mean ± SEM of four to eight experiments. *P<0.05 compared with vehicle control (veh, PBS) and ^†P<0.05 compared with EGF-treated cells.