Verapamil treatments overcome the cytotoxic effect of MTZ in zebrafish larvae. (A) Experimental timeline and treatment groups. Larvae (3 dpf) were randomly divided into five groups: Group 1 (untreated), normal developmental control; Group 2 (MTZ alone), larvae exposed to 10 mM MTZ for 24 h (to damage β-cells); Group 3 (Verapamil → MTZ), pre-treatment with 10 µM verapamil for 24 h before 10 mM MTZ exposure for 48 h; Group 4 (MTZ → Verapamil), post-treatment with 10 µM verapamil for 48 h after 10 mM MTZ insult for 24 h; Group 5 (MTZ + Verapamil), co-treatment with both 10 µM verapamil and 10 mM MTZ for 72 h. mCherry fluorescence reporter protein (ChFP) expressed in the larvae’s β-cells was imaged at 4 dpf (T0) and 6 dpf (T1). The change in ChFP intensity between T0 and T1 was measured. (B) Representative images of β-cells (red fluorescence) at T0 post-MTZ damage and T1 at recovery phase. Inserts show the magnified ChFP area. (C) Quantification of β-cell fluorescence (ChFP) at T1. Untreated Group 1 showed the physiological development of β-cells. The MTZ-alone group, Group 2, showed severe loss of fluorescence at T1, whereas the verapamil-treated groups 3–5 showed an improved β-cell preservation or recovery. Images were taken using Stereo Discovery 1.2 ZIESS microcopy. Experiments were performed in three independent trials (n = 20–30 larvae/group). Data are presented as mean ± SEM values. * p = 0.016, ** p = 0.01, *** p = 0.0004.
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