SiRNA Knockdown of Ribonucleotide Reductase Inhibits Melanoma Cell Line Proliferation Alone or Synergistically with Temozolomide Jonathan E. Zuckerman,

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siRNA Knockdown of Ribonucleotide Reductase Inhibits Melanoma Cell Line Proliferation Alone or Synergistically with Temozolomide Jonathan E. Zuckerman, Teli Hsueh, Richard C. Koya, Mark E. Davis, Antoni Ribas Journal of Investigative Dermatology Volume 131, Issue 2, Pages 453-460 (January 2011) DOI: 10.1038/jid.2010.310 Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Knockdown of ribonucleotide reductase subunit-2 (RRM2) mRNA and protein expression by small interfering RNA (siRNA) siR2B+5 in melanoma cells. (a) Quantitative real-time reverse transcription PCR analysis of RRM2 mRNA levels in HT-144 cells 48hours after transfection with 5nmoll−1 of RRM2-targeted siRNA (siR2B+5) or a non-targeting control (siCON) siRNA (error bars, n=3). (b) Quantification of RRM2 protein expression by western blot band densitometry analysis, average of three independent experiments; one blot is pictured (error bars, n=3). 5′-RNA ligand-mediated rapid amplification of complementary DNA ends detection of siRNA induced mRNA cleavage fragment HT-144 cells transfected using either (c) LipofectamineRNAiMax or (d) targeted cyclodextrin-containing polymer (CDP)/siRNA nanoparticles (arrows point to position of the predicted 209bp amplicon). (e) Sequencing chromatographs from positive rapid amplification of complementary DNA end bands. Journal of Investigative Dermatology 2011 131, 453-460DOI: (10.1038/jid.2010.310) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 In vitro antiproliferative effects of ribonucleotide reductase subunit-2 knockdown by siR2B+5 small interfering RNA (siRNA) in a panel of melanoma cell lines. (a) Bioluminescent-based viability assay on a panel of cell lines 72hours after transfection with 5nmoll−1 siRNAs, siR2B+5 (black columns), or siCON (white columns). Cell line oncogenic mutations are indicated by (*) NRAS codon 61 mutation, (+) BRAF V600E mutation, or (¶) NRAS/BRAF wild type. (b) MTS viability assay of M202 melanoma cells transfected with a range of siR2B+5 concentrations from 0.005 to 50nmoll−1 (black circles), or siCON 0.5–50nmoll−1 (white circles) performed at 48, 72, 96, and 120hours following transfection (only the 120-hour siCON time points are displayed). All cell viability data are normalized to untreated control samples (error bars, n=3). Journal of Investigative Dermatology 2011 131, 453-460DOI: (10.1038/jid.2010.310) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 G1/S-phase cell cycle arrest, but little apoptosis, is induced by siR2B+5 small interfering RNA ribonucleotide reductase subunit-2 knockdown. (a) Propidium idodide cell cycle analysis of HT-144 melanoma cells 48hours after treatment with 5nmoll−1 siR2B+5 or siCON; histograms are representative of at least three independent experiments. (b) Annexin-V-FITC/propidium iodide apoptosis analysis of HT-144 cells 72hours after treatment with 2.5 or 5nmoll−1 of siR2B+5, siCON, or staurosporin (SSP, positive control). Journal of Investigative Dermatology 2011 131, 453-460DOI: (10.1038/jid.2010.310) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Cell cycle arrest profiles vary between different melanoma cell lines, despite similar level of siR2B+5 small interfering RNA (siRNA) knockdown of ribonucleotide reductase subunit-2 expression. Propidium iodide cell cycle analysis of (a) M202 or (b) M207 melanoma cell lines 48hours after treatment with 5nmoll−1 siR2B+5 or siCON siRNA. Western blot analysis of (c) M202 or (d) M207 cells 48hours after transfection with 5nmoll−1 siR2B+5 siRNA in the presence of lipofectamine RNAiMax. Journal of Investigative Dermatology 2011 131, 453-460DOI: (10.1038/jid.2010.310) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Synergy between siR2B+5 and temozolomide treatment. Bioluminescence-based cell viability analysis of M202 cells (a) or HT-144 cells (b) 72hours after treatment with increasing doses of siR2B+5 (nmoll−1) (triangular boxes), temozolomide-sol (nmoll−1) (circular boxes), or co-treatment with both agents (square boxes). Data are normalized to an untreated sample. Synergy analysis of M202 (c) and HT-144 (d) cell viability data following co-treatment with siR2B+5 siRNA and temozolomide using the combination index (CI) method. CI values <1 indicate synergy. Journal of Investigative Dermatology 2011 131, 453-460DOI: (10.1038/jid.2010.310) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions