1. The Epigenetic Regulator I-BET151 Induces BIM-Dependent Apoptosis and Cell Cycle Arrest of Human Melanoma Cells 
Stuart J. Gallagher, Branka Mijatov, Dilini Gunatilake, Jessamy C. Tiffen, Kavitha Gowrishankar, Lei Jin, Gulietta M. Pupo, Carleen Cullinane, Rab K. Prinjha, Nicholas Smithers, Grant A. McArthur, Helen Rizos, Peter Hersey 
Journal of Investigative Dermatology 
Volume 134, Issue 11, Pages (November 2014)
DOI: /jid
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2. Figure 1
I-BET151 inhibits the growth of melanoma cellsin vitroandin vivoand induces cell death. (a) Me1007 melanoma cells were treated with 1, 10, 100 μM I-BET151, or vehicle control and cell growth was assessed over 72 hours using CellTiter Glo assay, with data normalized to a 0-h vehicle control. Error bars: SEM, n=8. (b) Average tumor volume of xenografted Patient-1-post cells in NOD/SCID mice treated with I-BET151 or vehicle control. (c) Cell death of matched pre/post primary lines, human dermal fibroblasts (HDF), human melanocytes (HEM), and continuous melanoma cell lines after 48 hours of 10 μM I-BET151 treatment was quantified by annexin-V staining. (d) The cell cycle distribution was assessed using flow cytometry. Results are from at least three individual experiments.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
I-BET151 increases BIM and induces caspase-dependent apoptosis. (a) The levels of apoptosis-related proteins were measured by western blotting melanoma cell lines treated with 10 μM I-BET151 for 48 hours. (b) Apoptosis was measured in cells pretreated with 10 μM of the pan-caspase inhibitor Q-VD-OPh and then treated for 48 hours with 10 μM I-BET151. TRAIL treatment was used as a positive control to activate caspase-dependent apoptosis in Mel-RM and Patient-3-pre cells. (c) Mitochondrial depolarization of cell lines treated with 10 μM I-BET151 for 48 hours was measured with JC-1 staining. (d) BIM mRNA was measured in cells by real-time RT-PCR after 6 hours of 10 μM I-BET151 treatment. (e) Apoptosis in cells after BIM was ablated using two separate siRNAs and cells were treated with 10 μM I-BET151 for 48 hours and (f) the efficiency of BIM knockdown was shown by western blotting. The panels showing BIML and BIMS are darker exposures of the same blot as BIMEL, to allow easier visualization of these bands. RT-PCR, reverse transcriptase–PCR; siRNA, small interfering RNA; TRAIL, tumor necrosis factor–related apoptosis-inducing ligand.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
I-BET151 induces p21-dependent cell cycle arrest. (a) The level of cell cycle regulators in continuous melanoma cells treated with 10 μM I-BET151 for 48 hours was measured by western blotting, and (b) in primary melanoma cell lines. (c) Cell cycle was measured in cells after p21 silencing and cells were treated with 10 μM I-BET151 for 48 hours. (d) The efficiency of the p21 knockdown was assessed by western blotting. Top panel images are from nonadjacent lanes of the same blot.
Journal of Investigative Dermatology  , DOI: ( /jid )
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5. Figure 4
Individual targeting of BET proteins causes cell cycle arrest and apoptosis. (a, b) BRD2, 3, and 4 were individually knocked down with siRNA in Me1007 and patient-3-pre cells and their levels assessed by real-time RT-PCR after 72 hours. (c, d) Levels of BIM and cell death were assessed by real-time RT-PCR and annexin-V staining, respectively. Levels are normalized to control siRNA-treated cells. (e, f) Levels of p21 and the percentage of cells in G1 phase were assessed by real-time RT-PCR and cell cycle analysis, respectively. Levels are normalized to control cells. *P<0.05. BET, bromodomain and extraterminal proteins; RT-PCR, reverse transcriptase–PCR; siRNA, small interfering RNA.
Journal of Investigative Dermatology  , DOI: ( /jid )
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6. Figure 5
Changes in expression of apoptosis-related genes. Expression level of apoptosis-related genes was determined by microarray after 6 or 24 hour I-BET151 treatment. Genes that were significantly changed collectively and with a greater than 50% median fold change (log2 FC>0.58) are presented. Red bars, proapoptotic genes. Green, antiapoptotic genes. Blue, both pro- and antiapoptotic functions described. Bars are log2 fold change compared with a DMSO-treated control at each time point. *Value exceeds axis maximum (see Supplementary Material online for actual value).
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions