Fig. 2.

Insulin induces DNA damage, ROS and Ca²⁺ homoeostasis imbalance. A, C Comet images assessing DNA damage in Ishikawa cells and RL-952 cells after treatment with insulin (100 nM) for 3 h. B, D Statistical analysis of tail DNA, tail length and comet length by comet assay software. n = 100. E Immunoblot of DNA damage repaired response kinase p-ATR and DNA damage marker γ-H2AX in Ishikawa cells after insulin (10, 100 nM) treatment for 0, 3 and 6 h. F Immunoblot of p-ATR, p-Chk1 and γ-H2AX in Ishikawa cells after insulin (100 nM) treatment for 0, 3, 6, 12 and 24 h. G Confocal microscopy detecting transient changes of mitochondrial Ca²⁺ after insulin (100 nM) stimulation. (200×) Ishikawa cells are loaded with mitochondrial probe Mito-tracker and mitochondrial Ca²⁺ probe Rhod-2, then stimulated by vehicle or insulin. H Fluorescence intensity indicating mitochondrial Ca²⁺ concentration change within 600 s treatment. Each curve indicates the change in a single isolated cell. I, J ROS analysis in Ishikawa cells after stimulation by vehicle or insulin (100 nM). CM-H2DCFDA is ROS indicator. K Immunoblot of γ-H2A in Ishikawa cells after insulin and/or NAC treatment. Histogram plot shows γ-H2A expression level relative to β-actin. *P < 0.05, insulin treatment alone compared with vehicle. #P < 0.05, NAC + insulin combination treatment compared with insulin treatment alone