Nb100 56113

The expression of Bcl-2, Bax and cleaved caspase-3 proteins were investigated by immunofluorescence method as previously described [54 (link)]. In brief, the 4T1 cells were seeded in a 6-well plate and exposed to the DE-EDCP and cisplatin at concentration of 31.25 μM for 24 hours. After washing the cells twice with PBS, they were fixed in 4% paraformaldehyde at 25°C for 20 min. The cells were stained with rabbit polyclonal antibody specific for Bcl-2 (sc-783, Santa Cruz Biotech. Inc CA, USA), Bax (sc-493, Santa Cruz Biotech. Inc CA, USA) and active/cleaved caspase-3 (NB100-56113, Novus Biologicals, UK). After incubation, the cells were washed and treated with appropriate secondary antibody, goat anti-rabbit IgG FITC (Ab6717-1, Abcam, Cambridge, United Kingdom). The sections were mounted with ProLong Gold antifade reagent with DAPI (Invitrogen) and analyzed at x 200 magnification using fluorescent microscope (Olympus BX 51).

The mouse xenograft tumors were fixed in paraformaldehyde for 1 h, followed by conventional paraffin‐embedding and sectioning (4 μm), and drying at 60℃. Paraffin sections were dewaxed with xylene and graded ethanol, and rinsed with distilled water. The slides were boiled in sodium citrate buffer (pH = 6.0, Solarbio, Beijing, China) for 10 min for antigen recovery. The sections were incubated in 3% hydrogen peroxide (Solarbio) for 10 min to block endogenous peroxidase activity and in 5% normal goat serum (ZGB‐BIO, Beijing, China) to block nonspecific protein binding sites. The sections were then incubated with antibodies against cleaved‐caspase‐3 (1:500; NB100‐56113, Novus Biologicals) and β‐Tubulin‐III (ab52623, RRID: AB_2819160, 1:800; Abcam) overnight at 4°C and subsequently incubated with goat antirabbit biotin‐conjugated secondary antibody (ab205718, 1:800; Abcam) for 0.5 h at room temperature. The reaction products were characterized using diaminobenzidine (Sigma‐Aldrich Chemical Company, St Louis, MO, USA). Nuclei were counted lightly with hematoxylin (Solarbio), and immunostaining was observed under a light microscopy (Olympus Optical Co., Ltd., Tokyo, Japan). The mean OD values of different sections were compared by Image‐pro Plus software.

BCL1 cells were seeded in a six-well plate and exposed to the Pt(S-pr-thiosal)2 (0.05 mg/mL) for 12 h. Cells were washed twice with phosphate-buffered saline and then fixed in 4% paraformaldehyde at 25 °C for 20 min. For cell staining, rabbit polyclonal antibody specific for Bcl-2 (sc-783, Santa Cruz Biotech Inc., Santa Cruz, CA, USA), Bax (sc-493, Santa Cruz Biotech Inc.), and phospho NF-κB (ab131109 Abcam, Cambridge, UK) and active/cleaved caspase 3 (NB100-56113, Novus Biologicals) were used. After incubation, the cells were washed and treated with appropriate secondary antibody, goat anti-rabbit IgG FITC (Ab6717-1, Abcam). The sections were mounted with ProLong Gold antifade reagent with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen, Carlsbad, CA, USA). For the cell analysis, fluorescent microscope (Olympus BX 51) was used at 200× magnification.