Figure 7

ErGPCR is involved in the 20E-induced increase in cytosolic Ca²⁺levels. (A) Suramin inhibited the 20E-induced Ca²⁺ increase (1 μM 20E, 1 mM calcium chloride, 50 μM suramin). F represents the fluorescence of cells after treatment, whereas F₀ denotes the average fluorescence of cells before treatment. Fluorescence was detected every 6 s for 360 s using a Laser Scan Confocal Microscope Carl Zeiss LSM 700 (Thornwood, NY, USA) at 555 nm and then analyzed using the Image Pro-Plus software. (B) ErGPCR knockdown using dsRNA (5 μg/mL) incubation inhibited the 20E-induced Ca²⁺ increase. (C) The T-type calcium channel blocker flunarizine dihydrochloride (FL, 50 μM) inhibited the 20E-indued Ca²⁺ influx. The L-type calcium channel blocker verapamil hydrochloride (Ve, 100 μM) did not affect the 20E-induced Ca²⁺ influx. (D) The transient receptor potential (TRP) channel blocker pyrazole (Pyr3, 10 μM) inhibited the 20E-induced Ca²⁺ increase. 2-Aminoethoxydiphenyl borate (2-APB) (10 μM to 100 μM) did not affect 20E-induced Ca²⁺ influx. (E) qRT-PCR showing the involvement of Ca²⁺ signal in 20E-induced gene expression. Cells were incubated in 1 μM 20E for 6 h after different inhibitors pretreatment for 1 h and the RNA was isolated for qRT-PCR. β-actin was used as the quantitative control. *indicates significant differences (p < 0.05) among the treatments via Student’s t test from three independent replicates. (F) Western blot showing the involvement of Ca²⁺ signal in 20E-induced Calponin phosphorylation. pIEx-4-Cal-GFP was overexpressed in HaEpi cells for 48 h. The cells were treated with 1 μM 20E for 1 h after different inhibitors pretreatment for 1 h. The GFP was overexpressed as controls.