Fig. 3
Regulation of AMPK signaling and TXNIP expression in BCa cells by 1,25(OH)2D3. a The AMPK-TXNIP signaling axis was found to be differentially regulated by a 72-h treatment with 1,25(OH)2D3 (100 nM). AMPK signaling was found to be induced and TXNIP expression reduced in MCF-7 cells in response to treatment. Western blot and densitometric analysis of TXNIP expression in MCF-7 cells treated for 24 (b) and 72 h (c) with 1,25(OH)2D3. Statistical significance between DMSO- and 1,25(OH)2D3-treated cells is calculated using a two-tailed Student’s t test, where p values less than or equal to 0.05, 0.01, and 0.001 are depicted in the figures by *, **, and ***, respectively. Error bars ± SD; n = 3. d–g 72 h treatment with 1,25(OH)2D3 significantly induces intracellular ROS levels. Microscopic analysis of cellular superoxide using DHE staining (d) and associated quantification (e). Hoechst dye was used to stain nuclei. Mitochondrial superoxide was stained using MitoSOX Red, and mitochondria were stained using MitoTracker Green (f). Microscopic images were taken every 15 min for 5 h, and fluorescence intensity of MitoSOX Red was quantified (g)