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. Author manuscript; available in PMC: 2021 Jul 26.
Published in final edited form as: Mol Cancer Ther. 2010 Dec;9(12):3289–3301. doi: 10.1158/1535-7163.MCT-10-0562

Figure 5.

Figure 5.

Effect of vorinostat (V) + flavopiridol (Fl) on expression of G2/M proteins. Representative neuroblastoma cell lines with wt TP53 (CHLA-136) and mt TP53 (CHLA-90) are shown. Cells were pre-treated with 2 μM vorinostat (V) for 24 hrs, and then 0.2 μM flavopiridol (Fl) was added for an additional 6, 9, 12, 16, 18, or 24 hrs. A. Immunoblot analysis of cyclin B1 (55 kDa), Cdk1 (34 kDa), and pCdk1 Thy15/Tyr15 (34 kDa) protein expression. Equal loading of protein was confirmed by β-actin expression. B. Immunoblot analysis of MPM2 (84 kDa), Mad2 (24 kDa), and Plk1 (62 kDa) protein expression. Equal loading of protein was confirmed by β-actin expression. C. Quantitative analysis of expression of cyclin B1, MPM2, Mad2, and Plk1 proteins as the ratio to β-actin in CHLA-136 (■) and CHLA-90 (●) cell lines treated with vorinostat + flavopiridol at 6, 9, 12, 16, 18, and 24 hrs. C – Vehicle treated controls, V – treatment with 2 μM vorinostat. D. CHLA-90 cells were transfected with 200 pMol total (100 pMol of target siRNA + 100 pMol scrambled siRNA for single targets and 100 pMol of each in co-transfection experiments) of scrambled, Mad2, cyclin B1, and Plk1 siRNAs. Forty eight hrs later, transfection efficacy was confirmed by immunoblotting (data not shown), and 72 hrs later cell clonogenicity was determined using the DIMSCAN assay. The DIMSCAN assay was performed in 3 separate experiments. Lanes: 1 – control; 2 – scrambled siRNA; 3 – Mad2 siRNA; 4 – cyclin B1; 5 – co-expression of Mad2 and cyclin B1 siRNAs; 6 – Plk1 siRNA; 7 - co-expression of Mad2 + Plk1 siRNA.