MCF-7 cells were grown on coverslips to 70–80% confluence, then fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.5% TritonX-100 for 10 min after 24 h. After blocking with 3% Albumin Bovine V (A8020, Solarbio, Beijing, China) for 1 h, the slides were quickly and gently washed with PBS. The cells were then incubated with the NQO1 antibody (1:500) at 4°C overnight, and followed by incubation

with Alexa Fluor® 568 goat anti-mouse IgG (H + L) (A11004, 1:1000, Invitrogen, Carlsbad, CA, USA) for 1 h. After washing with PBS, cells were counterstained with 49-6-diamidino-2-phenylindole (DAPI) (C1006, Beyotime, Shanghai, China) and the coverslips were mounted with Antifade Mounting Medium (P0126, Beyotime) [18]. Finally, the IF signals were visualized under Nutlin-3a in vivo a Leica SP5II CLSM microscope (Heidelberg, Germany) with filters for the corresponding fluorescent stains. Western blotting Fresh tissue samples were ground to powder in liquid nitrogen and lysed with SDS-PAGE sample buffer. Equal protein samples (20 μg) were separated on 10.5% SDS polyacrylamide gels and transferred to PVDF membranes (Immobilon P, Millipore, Bedford, MA, USA). Membranes were blocked with 5% fat-free milk in phosphate-buffered saline

with Tween-20 for 1 h at RT. Membranes were incubated with the NQO1 antibody (1:1000) overnight at 4°C, and then with horseradish peroxidase-conjugated goat anti-mouse IgG (CWBIO, China, CW0096A). NQO1 expression was detected using ECL Prime western blotting detection reagent (Amersham) Crenolanib according to the manufacturer’s instructions. Anti-β-actin mouse monoclonal antibody (CW0096A CWBIO, China) was used as a loading control [19]. Quantitative real-time PCR (qRT-PCR) As described previously [20], total RNA samples from eight of primary tumor materials were extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. selleck screening library The extracted RNA was pretreated with RNase-free DNase, and 2 μg RNA from each sample was used for cDNA synthesis primed with random hexamers. For the PCR amplification

of NQO1 cDNA, an initial BAY 73-4506 clinical trial amplification step using NQO1 specific primers was performed with denaturation at 95°C for 15 min, followed by 38 denaturation cycles at 95°C for 30 s, primer annealing at 60°C for 30 s, and a primer extension phase at 72°C for 30 s. Upon the completion of the cycling steps, a final extension step at 72°C for 7 min was conducted before the reaction mixture was stored at 4°C. Real-time PCR was then employed to determine the fold increase of NQO1 mRNA in each of the primary breast tumors relative to the paired adjacent non-tumor tissue taken from the same patient. Double-stranded DNA specific expression was tested by the comparative Ct method using 2-ΔΔCt. Primers were as follows: NQO1 5′-GGC AGA AGA GCA CTG ATC GTA-3′, and 5′-TGA TGG GAT TGA AGT TCA TGG C-3′; GAPDH 5′-CAT CAC CAT CTT CCA GGA GCG-3″, and 5′-TGA CCT TGC CCA CAG CCT TG-3′.