Changes in expression of cell cycle regulatory molecules and apoptosis-related genes during IL-6 treatment in M1-V1 and M1-G1.

(A) Northern blot analysis on expression of cell cycle regulatory molecules and apoptosis-related genes in M1-V1 and M1-G1 during the culture with recombinant human (rh) interleukin 6 (IL-6). M1-V1 and M1-G1 were cultured with 20 ng/mL of rhIL-6, and total cellular RNA was isolated at the time indicated. Fifteen μg of each sample was electrophoresed on formaldehyde agarose gels, blotted onto the filters, and hybridized with ³²P-labeled probes as indicated. (B) Western blot analysis on cyclin D1 expression (upper panel) and a cdk4-associated immune complex kinase assay (lower panel) in M1-V1 and M1-G1 during the culture with rhIL-6. Total cellular lysates (15 μg per each lane) obtained from the cultured cells were subjected to SDS-PAGE and probed with rabbit anti-cyclin D1 monoclonal antibody. Immunoreactive proteins were visualized with the enhanced chemiluminescence detection system (upper panel). cdk4 was immunoprecipitated from equal amounts of the cell lysates prepared from the cultured cells. Immune complex kinase assay was performed in kinase buffer containing 5 μg of GST-Rb fusion protein and 20 μCi of γ-[³²P]ATP for 30 minutes at 30°C. After the addition of a protein-loading buffer, samples were subjected to SDS-PAGE. The gels were stained with Coomassie blue, destained, dried, and subjected to autoradiography (lower panel). (C) Western blot analysis on bcl-2 expression in M1-V1 and M1-G1 during the culture with rhIL-6. Fifteen μg of total cellular lysates obtained from the cultured cells were subjected to SDS-PAGE and probed with rabbit anti-bcl-2 polyclonal antibody.