Cells are washed with ice-cold PBS containing 0.1 mM sodium orthovanadate, and total proteins are isolated using RIPA lysis buffer, which includes protease inhibitors (leupeptin, antipain, and aprotinin), 0.5 mM PMSF, and 0.2 mM sodium orthovanadate. Protein amounts are quantified using the Bio-Rad protein assay. Equal amounts of proteins are loaded onto an sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel, transferred onto nitrocellulose membrane, and probed with the antibody of interest: mouse monoclonal c-Myc and mouse monoclonal p21 antibodies; rabbit polyclonal phospho-Histone H2A.X, rabbit polyclonal acetyl-Histone H3 (Lys9), and rabbit polyclonal acetyl-Histone H4 (Lys8) antibodies; mouse monoclonal p27/KIP1 antibody; mouse monoclonal anti-β-actin; and mouse monoclonal anti-GADD45G. Membranes are then washed, reprobed with appropriate horseradish peroxidase-conjugated secondary antibodies, and developed with SuperSignal chemiluminescent substrate.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

AE17 and TC-1 cancer cells (1×10⁶ cells) are injected into the flanks of adult female C57Bl/6 mice and severe combined immunodeficiency (SCID) mice. M30 (10×10⁶ cells), A549 (5×10⁶ cells), H69 (2.5×10⁶ cells), BK-T (6.5×10⁶), H526 (10×10⁶), and RG1 (10×10⁶) cells are also injected, but in the presence of matrigel, into the flanks of SCID mice. When tumors reach 100 to 500 mm³, panobinostat is administered via i.p. injections (10-20 mg/kg) on a daily schedule (5-days-on, 2-days-off regimen) for the entire duration of the experiment. Control micereceive i.p. injections with dextrose 5% in water. Every tumor is measured with a caliper at least twice weekly. For evaluation of the effects of combination therapy on SCLC-derived tumors, SCID mice with H69 tumors are administered panobinostat. Three days after the initiation of panobinostat, and again 1 wk later, etoposide (40 mg/kg) is administered i.p.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

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