Fig. 2: Attenuation of zinc-induced neuronal death by EGF treatment in mouse cerebrocortical cultures.

A Microscopic images (left) and a quantitative graph (right) depict propidium iodide (PI)-stained damaged neuronal cells at 20 hours after exposure to 40 µM zinc, with or without EGF (100 ng/ml). Scale bar: 100 µm. The bar graph indicates the fluorescence intensity of PI measured using the Image J program (mean ± SEM, n = 12 different fields taken from ≥4 independent biological replicate experiments, ^####p < 0.0001 for control (CTL), and ***p < 0.001 for zinc exposure, analyzed using ANOVA with Dunnett’s post-hoc test). B Bars represent LDH release at 20 h after exposure to zinc (40 µM) with or without EGF (100 ng/ml) (mean ± SEM, n = 5 independent biological replicate experiments, **p < 0.01; two-tailed Student’s t-test). C Bars represent LDH release at 20 h after exposure to zinc (40 µM) with or without EGF (100 ng/ml) pretreatment for the indicated time points (mean ± SEM, n = 4 independent biological replicate experiments, *p < 0.05 or ***p < 0.001 for zinc exposure, analyzed using ANOVA with Dunnett’s post-hoc test). D Microscopic images (left) showing GFP fluorescence following transient transfection with pCMV3-untagged negative control vector (NC) or GFP-EGFR tagged plasmid. Scale bar: 75 µm. The right bar graph represents LDH release at 18 h after exposure to zinc (80 µM) in empty vector (NC) or EGFR overexpressing HEK cells (mean ± SEM, n = 4 independent biological replicate experiments, ****p < 0.0001; two-tailed Student’s t-test). E Microscopic images (left) and a quantitative graph (right) show PI-stained damaged neuronal cells at 20 hours after exposure to 40 µM zinc, with or without leupeptin (Leu; 100 µM), CA074 (20 µM), or TPEN (1 µM). Scale bar: 200 µm. Bars represent the fluorescence intensity of PI (mean ± SEM, n = 14 different fields taken from ≥4 independent biological replicate experiments, ^####p < 0.0001 for control (CTL), and **p < 0.01 or ****p < 0.0001 for zinc exposure, analyzed using ANOVA with Dunnett’s post-hoc test). F Bars indicate LDH release at 20 h after exposure to zinc (40 µM) with or without leupeptin (Leu; 100 µM), CA074 (20 µM), or TPEN (1 µM) (mean ± SEM, n = 6 or 16 taken from ≥4 independent biological replicate experiments, **p < 0.01 or ****p < 0.0001 for zinc exposure, analyzed using ANOVA with Dunnett’s post-hoc test). G Bars represent LDH release at 20 h after exposure to zinc (40 µM) with or without EGF (100 ng/ml), CA074 (20 µM), or EGF plus CA074 (mean ± SEM, n = 7 independent biological replicate experiments, **p < 0.01 or ***p < 0.001 for zinc exposure, analyzed using ANOVA with Dunnett’s post-hoc test). No significant difference (ns) was observed among treatment with EGF, CA074, or EGF plus CA074.