Immunofluorescence microscopy for PUMA expression MCF-7 stably transfected with Mito-GFP were maintained in media supplemented with 400 mg/mL G418. The cells were plated on cover slips and treated with DMSO (control) or 5 mM BITC for 24 h followed by fixing in paraformaldehyde. Cells were permeabilized with Triton X-100 and incubated with blocking buffer containing bovine serum albumin in phosphate-buffered saline (PBS) for 1 h at room temperature. Cells were then incubated with anti-PUMA antibody in blocking buffer overnight at 4uC. Cells were stained with Alexa Fluor 568-conjugated secondary antibody and DAPI prior to mounting. Immunofluo- rescence was examined under a Leica DC300F fluorescence microscope. DAPI assay DAPI staining was done to quantify apoptotic cells with condensed and fragmented DNA. The DMSO-treated control and BITC-treated cells were fixed in paraformaldehyde and permeabilized with 0.4% Triton X-100. 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