Anti cyclin b1

Bone marrow cells from untreated animals were collected as described above. First, we measured the expression of FPR2 in LSK cells. The anti-FPR2-FITC antibody was used (Bioss, Massachusetts, USA) to quantify this expression by flow cytometry. Afterwards, bone marrow cells were treated with rAnxA1 (100 nM) for 12 h, and viability of the LSK population determined using 7-aminoactinomycinD (7-AAD; Sigma Aldrich, USA); cell cycle phases and expression of Ki67 and Notch-1 were also evaluated. These quantifications were carried out by flow cytometry. Cells were fixed in 2% paraformaldehyde for 30 min, washed with 0.1 M glycine, and permeabilized with 0.001% Triton X-100. Subsequently, 2 × 10⁶ cells were incubated with primary anti-cyclin B1, anti-Notch-1 or anti-Ki67 (Cell Signaling Technology) antibodies for 2 h, and then 40 min with secondary rabbit Anti-IgG Alexa Fluor 488 (Molecular Probes/Invitrogen, USA). Analyses were performed on the LSK population gated as described above, using an Accuri C6 flow cytometer (Becton Dickinson, USA) and the FlowJo software.

Immunohistochemistry was carried out using formalin-fixed paraffin-embedded (FFPE) sections as described [28 (link)]. The sections were blocked in phosphate buffered saline-tween 20 (PBST) using 10% normal goat serum. The primary antibodies were diluted in PBST containing 5% goat serum. The primary antibodies used were: anti-AFP (Santa Cruz; rabbit polyclonal; 1:50); anti-CD31 (Dako; mouse monoclonal; 1:50); anti-PCNA (Cell Signaling; mouse monoclonal; 1:100); anti-OPN (Millipore; rabbit polyclonal; 1:500); anti-CyclinB1 (1:100, rabbit polyclonal; Cell Signaling). Secondary antibodies were diluted in PBST containing corresponding 2.5% blocking serum. The signals were developed by avidin-biotin-peroxidase complexes with a DAB substrate solution (Vector laboratories). Images were analyzed using an Olympus microscope.

The cells were treated with ATF₂₄-PEG-Lipo-β-E and/or DDP for the indicated times. The PVDF membranes were incubated with anti-cyclin B1 (1:1,000, Cell Signaling Technology, anti-Bcl-2 (1:1,000; Cell Signaling Technology), anti-Bax (1:1,000; Cell Signaling Technology), anti-cleaved PARP (1:1,000; Cell Signaling Technology), anti-Cdc25C (1:200; Santa Cruz Biotechnology, Dallas, TX, USA), anti-Cdc2 p34 (1:200; Santa Cruz Biotechnology), anti-cleaved caspase-3 (1:400; Abcam, Cambridge, UK), and anti-GAPDH (1:1,000; Cell Signaling Technology) primary antibodies at 4 °C overnight. The other steps were the same as described above³⁸ .

Anti‐mortalin (#3593), anti‐p‐Erk1/2 (#4370), anti‐p‐Jun N‐terminal kinase (JNK; #4668), anti‐JNK (#9252), anti‐c‐Raf (#9421), anti‐p‐c‐Raf (#9422), anti‐Poly ADP ribose polymerase (PARP) (#9542), anti‐Cyclin‐D1 (#2978) and anti‐Cyclin‐B1 (#12231) were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti‐C‐myc (sc‐764) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti‐β‐actin was purchased from Sigma‐Aldrich (St. Louis, MO, USA). Anti‐Erk1/2 (KC‐5E01) was purchased from Kang‐Chen Bio‐Tech, Inc. (Shanghai, China). Goat antimouse secondary antibodies (115‐035‐003) and goat anti‐rabbit secondary antibodies (111‐035‐003) were obtained from Jackson ImmunoResearch Laboratories, (West Grove, PA, USA).

Western blotting was performed according to protocol. Briefly, cells were lysed in RIPA buffer. Cellular proteins were collected and subjected to 10% SDS-PAGE and transferred onto PVDF membranes. The membranes were then incubated with specific primary antibodies (anti-cyclin B1 (#12231, Cell Signaling Technology, USA), anti-cyclin D1(#55506, Cell Signaling Technology, USA), anti-cyclin E1 (#20808, Cell Signaling Technology, USA), anti-N-cadherin (#13116, Cell Signaling Technology, USA), anti-vimentin (#5741, Cell Signaling Technology, USA), anti-TK1 (#28755, Cell Signaling Technology, USA), and anti-GAPDH (#2118, Cell Signaling Technology, USA)) overnight at 4°C. After the membranes were incubated with secondary antibodies, they were subjected to immunoblot analysis using an ECL immunoblotting kit according to the manufacturer's protocol.