Fig. 4

Genipin enhances mitochondria dysfunction through Mcl-1. a–c OCR was measured in AGS cells treated with Genipin using an XF analyzer. OCR levels were detected after addition of 2 μg/mL oligomycin, 5 μM m-chlorophenyl hydrazome, and 2 μM rotenone (a). The basal respiration OCR (b) and spare respiratory capacity (c) were quantified by OCR level. d, e AGS cells were treated with 150 μM Genipin for 24 h. Treated cells were stained with 10 μM TMRE (d) and 10 μM NAO (e) for 10 min at 37 °C. Stained cells were harvested and measured by flow cytometry. Fluorescence staining intensity was measured using flow cytometry. f, g 150 μM Genipin treated or untreated cells for 24 h were immunostained with Mitotracker (red) (f) and JC-1 (g). Images were captured using a confocal microscope. h Western blot analysis of mitochondrial electron transport chain proteins after Genipin treatment in AGS cells. i AGS cells were transfected with siMcl-1. Transfected cells were treated Genipin 150 μM for 24 h. MCl-1, VDAC, and SDHA protein levels were detected by western blotting with β-actin used as a loading control. j AGS cells were transfected with His-Mcl-1 plasmid. Mcl-1 overexpression cells were treated with 150 μM Genipin. Whole cell lysates were collected and incubated with the indicated antibodies