Fig. 4.

CaM1 and L-VGCC-mediated Ca²⁺ signalling pathways were activated by scoparone. a Expression of recombinant CaM1 protein (lane 1) by E. coli. b, c IC₅₀ of TFP (b) and EC₅₀ of scoparone (c) as determined by CaM1 protein assay. d, e The specific activation of CaM in T. cinnabarinus exposed to scoparone (d) and 30.4 μM TFP (IC₅₀, e) was determined. f, g Effects of scoparone on intracellular free Ca²⁺ levels ([Ca²⁺]i) in Sf9 cells. Positive staining for Ca²⁺ is shown as green zones in the images captured under a microscope. ‘LC₅₀ + TFP’ indicates TFP-pretreated (IC₅₀ dose of TFP) Sf9 cells incubated with 0.28 mg/mL scoparone for 48 h. h, i [Ca²⁺]i of GFP- and CaM-expressing cells exposed to 0.28 mg/mL scoparone for 48 h. j Specific induction of PDE activity against the substrate cAMP by recombinant CaM1 expressed by E. coli. k Cytotoxicity of scoparone towards CaM- and GFP-expressing cells; scale bar = 40 μm. l, m, n Representative whole-cell responses to L-VGCC agonism and channel activity were induced by scoparone in non-pretreated cells (l, n) and cells pretreated with 0.61 μM CaM1 (K_df dose of CaM1) (m, n). o, p, q Representative whole-cell responses to L-VGCC agonism and channel activity were induced by CaM1 in non-pretreated cells (o, q) and cells pretreated with 5.54 µM scoparone (EC₅₀ dose of scoparone) (p, q). The data are shown as the mean ± SD from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.