Lack of retinoblastoma proteins (Rb) continues to be correlated with level of resistance to PD0332991 (29), however, every one of the melanoma cell lines tested expressed detectable degrees of Rb. from 0.03 to 0.22 M. Fascaplysin inhibited clonogenic development and induced apoptosis also. Awareness to PD0332991, a therapeutic SNX-2112 CDK4/6 inhibitor was evaluated in the melanoma cell lines also. PD0332991 IC50 beliefs ranged from 0.13 to 2.29 M. Comparable to fascaplysin, PD0332991 inhibited clonogenic development of melanoma cells and induced apoptosis. Higher degrees of CDK4 proteins correlated with lower awareness to PD0332991 in the cell lines. Mixed treatment with PD0332991 as well as the BRAF inhibitor PLX4032, demonstrated additive anti-proliferative results in the BRAF mutant cell series Malme-3M. In conclusion, concentrating on CDK4 inhibits development and induces apoptosis in melanoma cells (11) showed p16INK4a mutation, promoter absence or methylation of appearance happened in 16, 25 and 82% of melanoma metastases, respectively. The p16INK4a proteins binds to CDK4/6 and inhibits connections with D-type cyclins, which would stimulate passage through the G1 phase from the cell cycle otherwise. The frequent lack of p16INK4a in melanomas shows that CDK4 activity could be unchecked in melanoma and could are likely involved to advertise uncontrolled proliferation of melanoma cells. Furthermore, overexpression or mutation of CDK4, coupled with amplification of cyclin D1, continues to be implicated in level of resistance to BRAF inhibition in V600E-mutated melanoma cells, and amplification of cyclin D1 is normally discovered in ~17% of BRAF V600E-mutated individual metastatic melanomas (12). The druggable character of kinases provides sparked considerable curiosity about seeking CDKs as novel goals in anticancer medication advancement. Selective inhibition of CDKs may limit the development of the tumour cell through the cell routine and facilitate the induction of apoptosis (6,13). Strategies and Components Cells and reagents Malme-3M, Sk-Mel-2, Sk-Mel-5, Sk-Mel-28, M14 and Lox-IMVI melanoma cell lines had been extracted from the Section of Developmental Therapeutics, Country wide Cancers Institute (Bethesda, MD, USA). WM-115 and WM-266-4 melanoma cell SNX-2112 lines had been extracted from the Western european Association Lifestyle Collection (UK). Malme-3M, Sk-Mel-2, Sk-Mel-5, Sk-Mel-28, M14 and Lox-IMVI cell lines had been preserved at 37C with 5% CO2 in RPMI-1640 moderate (Sigma-Aldrich, Co. Wicklow, Ireland) with 10% fetal leg serum (FCS; Lonza, Tewkesbury, UK). WM-115 and WM-266-4 had been preserved at 37C with 5% CO2 in minimal important moderate (MEM; Sigma-Aldrich) with 10% FCS (BioWhittaker, Walkersville, MD, USA), 2 mM L-glutamine (Lifestyle Technology, Dublin, Ireland), 1 mM nonessential proteins (Life Technology) and 1 mM sodium pyruvate (Lifestyle Technologies). Share solutions of fascaplysin (Merck Millipore, Watford, UK) (10 mM), PLX4032 (Sequoia Analysis Items Ltd., Pangbourne, UK) (10 mM), elacridar (Sigma-Aldrich) (10 mM) and temozolomide (Sigma-Aldrich) (103 mM) had been ready in dimethyl sulfoxide (DMSO) PD0332991 (supplied by Pfizer, Peapack, NJ, USA) (10 mM) was ready in ultrapure drinking water. InhibitorSelect? 384-well proteins kinase inhibitor collection I The InhibitorSelect proteins kinase inhibitor collection I (Merck Millipore) was given 160 proteins kinase inhibitors within a 384-well dish at a level of 25 l and a focus of 10 mM in DMSO and had been kept at ?80C. Share solutions (1 mM) had been made by dilution in DMSO, and kept at ?20C. Preliminary screening from the 160 proteins kinase inhibitors was performed at 1 M focus on the Sk-Mel-2 and Sk-Mel-28 cell lines. Cells/well (1103) had been seeded in 96-well plates. Plates had been incubated right away at 37C accompanied by addition of medications at the correct concentrations and incubated for an additional 5 times until wells had been 80C90% confiuent. At conclusion of the assay the colorimetric acidity phosphatase assay was utilized to determine cell viability. Proliferation assays and acidity phosphatase assay All cells lines had been seeded at 1103 cells/well in 96-well plates aside from Malme-3M and WM-115 that have been seeded at 2103 cells/well. Plates had been incubated right away at 37C accompanied by addition of medication at the correct concentrations and incubated for an additional 5 times until wells had been 80C90% confluent. All mass media had been removed as well as the wells had been cleaned once with phosphate-buffered saline (PBS; Sigma-Aldrich). Paranitrophenol phosphate substrate (7.25 mM; Sigma-Aldrich) in 0.1 M sodium acetate buffer with 0.1% Triton-X (Sigma-Aldrich) pH 5.5 was put into each well and incubated at 37C for 2 h. To avoid the response 50 l of just one 1 M NaOH was added as well as the absorbance was browse at 405 nM (guide, 620 nM). Clonogenic assays Malme-3M were seeded at 600 Sk-Mel-2 and cells/very well were seeded at 125 cells/very well within a 24-very well dish. The cells were incubated at 37C overnight. Media had been removed as well as the medications had been added at the correct concentrations. Drug formulated with medium was taken out after 4 times and changed with drug-free mass media. The moderate was thereafter replenished every 4/5 times. Malme-3M cells had been incubated for 17 times and Sk-Mel-2 cells had been incubated for two weeks. When prepared to stain, mass media were removed as well as the cells washed with PBS twice gently. The cells had been then set in frosty Methacare option (4C, 75% v/v methanol, 25% v/v acetic acid solution) for 30 min. The Methacare option.Furthermore, mutation or overexpression of CDK4, coupled with amplification of cyclin D1, continues to be implicated in level of resistance to BRAF inhibition in V600E-mutated melanoma cells, and amplification of cyclin D1 is detected in ~17% of BRAF V600E-mutated individual metastatic melanomas (12). The druggable nature of kinases has sparked considerable curiosity about pursuing CDKs as novel targets in anticancer medication advancement. inhibited clonogenic development and induced apoptosis. Awareness to PD0332991, a healing CDK4/6 inhibitor was also examined in the melanoma cell lines. PD0332991 IC50 beliefs ranged from 0.13 to 2.29 M. Comparable to fascaplysin, PD0332991 inhibited clonogenic development of melanoma cells and induced apoptosis. Higher degrees of CDK4 proteins correlated with lower awareness to PD0332991 in the cell lines. Mixed treatment with PD0332991 as well as the BRAF inhibitor PLX4032, demonstrated additive anti-proliferative results in the BRAF mutant cell series Malme-3M. In conclusion, targeting CDK4 inhibits growth and induces apoptosis in melanoma cells (11) demonstrated p16INK4a mutation, promoter methylation or lack of expression occurred in 16, 25 and 82% of melanoma metastases, respectively. The p16INK4a protein binds to CDK4/6 and inhibits interaction with D-type cyclins, which would otherwise stimulate passage through the G1 phase of the cell cycle. The frequent loss of p16INK4a in melanomas suggests that CDK4 activity may be unchecked in melanoma and may play a role in promoting uncontrolled proliferation of melanoma cells. Furthermore, mutation or overexpression of CDK4, combined with amplification of cyclin D1, has been implicated in resistance to BRAF inhibition in V600E-mutated melanoma cells, and amplification of cyclin D1 is detected in ~17% of BRAF V600E-mutated human metastatic melanomas (12). The druggable nature of kinases has sparked considerable interest in pursuing CDKs as novel targets in anticancer drug development. Selective inhibition of CDKs may limit the progression of a tumour cell through the cell cycle and facilitate the Rabbit Polyclonal to U12 induction of apoptosis (6,13). Materials and methods Cells and reagents Malme-3M, Sk-Mel-2, Sk-Mel-5, Sk-Mel-28, M14 and Lox-IMVI melanoma cell lines were obtained from the Department of Developmental Therapeutics, National Cancer Institute (Bethesda, MD, USA). WM-115 and WM-266-4 melanoma cell lines were obtained from the European Association Culture Collection (UK). Malme-3M, Sk-Mel-2, Sk-Mel-5, Sk-Mel-28, M14 and Lox-IMVI cell lines were maintained at 37C with 5% CO2 in RPMI-1640 medium (Sigma-Aldrich, Co. Wicklow, Ireland) with 10% fetal calf serum (FCS; Lonza, Tewkesbury, UK). WM-115 and WM-266-4 were maintained at 37C with 5% CO2 in minimal essential medium (MEM; Sigma-Aldrich) with 10% FCS (BioWhittaker, Walkersville, MD, USA), 2 mM L-glutamine (Life Technologies, Dublin, Ireland), 1 mM non-essential amino acids (Life Technologies) and 1 mM sodium pyruvate (Life Technologies). Stock solutions of fascaplysin (Merck Millipore, Watford, UK) (10 mM), PLX4032 (Sequoia Research Products Ltd., Pangbourne, UK) (10 mM), elacridar (Sigma-Aldrich) (10 mM) and temozolomide (Sigma-Aldrich) (103 mM) were prepared in dimethyl sulfoxide (DMSO) PD0332991 (provided by Pfizer, Peapack, NJ, USA) (10 mM) was prepared in ultrapure water. InhibitorSelect? 384-well protein kinase inhibitor library I The InhibitorSelect protein kinase inhibitor library I (Merck Millipore) was supplied with 160 protein kinase inhibitors in a 384-well plate at a volume of 25 l and a concentration of 10 mM in DMSO and were stored at ?80C. Stock solutions (1 mM) were prepared by dilution in DMSO, and stored at ?20C. Initial screening of the 160 protein kinase inhibitors was performed at 1 M concentration on the Sk-Mel-2 and Sk-Mel-28 cell lines. Cells/well (1103) were seeded in 96-well plates. Plates were incubated overnight at 37C followed by addition of drugs at the appropriate concentrations and incubated for a further 5 days until wells were 80C90% confiuent. At completion of the assay the colorimetric acid phosphatase assay was used to determine cell viability. Proliferation assays and acid phosphatase assay All cells lines were seeded at 1103 cells/well in 96-well plates except for Malme-3M and WM-115 which were seeded at 2103 cells/well. Plates were incubated overnight at 37C followed by addition of drug at the appropriate concentrations and incubated for a further 5 days until wells were 80C90% confluent. All media were removed and the wells were washed once with phosphate-buffered saline (PBS; Sigma-Aldrich). Paranitrophenol phosphate substrate (7.25 mM; Sigma-Aldrich) in 0.1 M sodium acetate buffer with 0.1% Triton-X (Sigma-Aldrich) pH 5.5 was added to each well and incubated at 37C for 2 h. To stop the reaction 50 l of 1 1 M NaOH was added and the absorbance was read at 405 nM (reference, 620 nM). Clonogenic assays Malme-3M were seeded at 600 cells/well and Sk-Mel-2 were seeded at 125 cells/well in a 24-well plate. The cells were incubated overnight at 37C. Media were removed and the drugs were added at the appropriate concentrations. Drug containing medium was removed after 4 days and replaced with drug-free media. The medium was replenished every 4/5 days thereafter. Malme-3M cells were incubated for 17 days.WM-115 and WM-266-4 were maintained at 37C with 5% CO2 in minimal essential medium (MEM; Sigma-Aldrich) with 10% FCS (BioWhittaker, Walkersville, MD, USA), 2 mM L-glutamine (Life Technologies, Dublin, Ireland), 1 mM non-essential amino acids (Life Technologies) and 1 mM sodium pyruvate (Life Technologies). inhibited clonogenic growth of melanoma cells and induced apoptosis. Higher levels of CDK4 protein correlated with lower sensitivity to PD0332991 in the cell lines. Combined treatment with PD0332991 and the BRAF inhibitor PLX4032, showed additive anti-proliferative effects in the BRAF mutant cell line Malme-3M. In summary, targeting CDK4 inhibits growth and induces apoptosis in melanoma cells (11) demonstrated p16INK4a mutation, promoter methylation or lack of expression occurred in 16, 25 and 82% of melanoma metastases, respectively. The p16INK4a protein binds to CDK4/6 and inhibits interaction with D-type cyclins, which would otherwise stimulate passage through the G1 phase of the cell cycle. The frequent loss of p16INK4a in melanomas suggests that CDK4 activity may be unchecked in melanoma and may play a role in promoting uncontrolled SNX-2112 proliferation of melanoma cells. Furthermore, mutation or overexpression of CDK4, combined with amplification of cyclin D1, has been implicated in resistance to BRAF inhibition in V600E-mutated melanoma cells, and amplification of cyclin D1 is recognized in ~17% of BRAF V600E-mutated human being metastatic melanomas (12). The druggable nature of kinases offers sparked considerable desire for going after CDKs as novel focuses on in anticancer drug development. Selective inhibition of CDKs may limit the progression of a tumour cell through the cell cycle and facilitate the induction of apoptosis (6,13). Materials and methods Cells and reagents Malme-3M, Sk-Mel-2, Sk-Mel-5, Sk-Mel-28, M14 and Lox-IMVI melanoma cell lines were from the Division of Developmental Therapeutics, National Tumor Institute (Bethesda, MD, USA). WM-115 and WM-266-4 melanoma cell lines were from the Western Association Tradition Collection (UK). Malme-3M, Sk-Mel-2, Sk-Mel-5, Sk-Mel-28, M14 and Lox-IMVI cell lines were managed at 37C with 5% CO2 in RPMI-1640 medium (Sigma-Aldrich, Co. Wicklow, Ireland) with 10% fetal calf serum (FCS; Lonza, Tewkesbury, UK). WM-115 and WM-266-4 were managed at 37C with 5% CO2 in minimal essential medium (MEM; Sigma-Aldrich) with 10% FCS (BioWhittaker, Walkersville, MD, USA), 2 mM L-glutamine (Existence Systems, Dublin, Ireland), 1 mM non-essential amino acids (Life Systems) and 1 mM sodium pyruvate (Existence Technologies). Stock solutions of fascaplysin (Merck Millipore, Watford, UK) (10 mM), PLX4032 (Sequoia Study Products Ltd., Pangbourne, UK) (10 mM), elacridar (Sigma-Aldrich) (10 mM) and temozolomide (Sigma-Aldrich) (103 mM) were prepared in dimethyl sulfoxide (DMSO) PD0332991 (provided by Pfizer, Peapack, NJ, USA) (10 mM) was prepared in ultrapure water. InhibitorSelect? 384-well protein kinase inhibitor library I The InhibitorSelect protein kinase inhibitor library I (Merck Millipore) was supplied with 160 protein kinase inhibitors inside a 384-well plate at a volume of 25 l and a concentration of 10 mM in DMSO and were stored at ?80C. Stock solutions (1 mM) were prepared by dilution in DMSO, and stored at ?20C. Initial screening of the 160 protein kinase inhibitors was performed at 1 M concentration on the Sk-Mel-2 and Sk-Mel-28 cell lines. Cells/well (1103) were seeded in 96-well plates. Plates were incubated over night at 37C followed by addition of medicines at the appropriate concentrations and incubated for a further 5 days until wells were 80C90% confiuent. At completion of the assay the colorimetric acid phosphatase assay was used to determine cell viability. Proliferation assays and acid phosphatase assay All cells lines were seeded at 1103 cells/well in 96-well plates except for Malme-3M and WM-115 which were seeded at 2103 cells/well. Plates were incubated over night at 37C followed by addition of drug at the appropriate concentrations and incubated for a further 5 days until wells were 80C90% confluent. All press were removed and the wells were washed once with phosphate-buffered saline (PBS; Sigma-Aldrich). Paranitrophenol phosphate substrate (7.25 mM; Sigma-Aldrich) in 0.1 M sodium acetate buffer with 0.1% Triton-X (Sigma-Aldrich) pH 5.5 was added to each well and incubated at 37C for 2 h. To stop the reaction 50 l of 1 1 M NaOH was added and the absorbance was go through at 405 nM (research, 620 nM). Clonogenic assays Malme-3M were seeded at 600 cells/well and Sk-Mel-2 were seeded at 125 cells/well inside a 24-well plate. The cells were incubated over night at 37C..Press were removed and the medicines were added at the appropriate concentrations. in the melanoma cell lines. PD0332991 IC50 ideals ranged from 0.13 to 2.29 M. Much like fascaplysin, PD0332991 inhibited clonogenic growth of melanoma cells and induced apoptosis. Higher levels of CDK4 protein correlated with lower level of sensitivity to PD0332991 in the cell lines. Combined treatment with PD0332991 and the BRAF inhibitor PLX4032, showed additive anti-proliferative effects in the BRAF mutant cell collection Malme-3M. In summary, targeting CDK4 inhibits growth and induces apoptosis in melanoma cells (11) exhibited p16INK4a mutation, promoter methylation or lack of expression occurred in 16, 25 and 82% of melanoma metastases, respectively. The p16INK4a protein binds to CDK4/6 and inhibits conversation with D-type cyclins, which would normally stimulate passage through the G1 phase of the cell cycle. The frequent loss of p16INK4a in melanomas suggests that CDK4 activity may be unchecked in melanoma and may play a role in promoting uncontrolled proliferation of melanoma cells. Furthermore, mutation or overexpression of CDK4, combined with amplification of cyclin D1, has been implicated in resistance to BRAF inhibition in V600E-mutated melanoma cells, and amplification of cyclin D1 is usually detected in ~17% of BRAF V600E-mutated human metastatic melanomas (12). The druggable nature of kinases has sparked considerable desire for pursuing CDKs as novel targets in anticancer drug development. Selective inhibition of CDKs may limit the progression of a tumour cell through the cell cycle and facilitate the induction of apoptosis (6,13). Materials and methods Cells and reagents Malme-3M, Sk-Mel-2, Sk-Mel-5, Sk-Mel-28, M14 and Lox-IMVI melanoma cell lines were obtained from the Department of Developmental Therapeutics, National Malignancy Institute (Bethesda, MD, USA). WM-115 and WM-266-4 melanoma cell lines were obtained from the European Association Culture Collection (UK). Malme-3M, Sk-Mel-2, Sk-Mel-5, Sk-Mel-28, M14 and Lox-IMVI cell lines were managed at 37C with 5% CO2 in RPMI-1640 medium (Sigma-Aldrich, Co. Wicklow, Ireland) with 10% fetal calf serum (FCS; Lonza, Tewkesbury, UK). WM-115 and WM-266-4 were managed at 37C with 5% CO2 in minimal essential medium (MEM; Sigma-Aldrich) with 10% FCS (BioWhittaker, Walkersville, MD, USA), 2 mM L-glutamine (Life Technologies, Dublin, Ireland), 1 mM non-essential amino acids (Life Technologies) and 1 mM sodium pyruvate (Life Technologies). Stock solutions of fascaplysin (Merck Millipore, Watford, UK) SNX-2112 (10 mM), PLX4032 (Sequoia Research Products Ltd., Pangbourne, UK) (10 mM), elacridar (Sigma-Aldrich) (10 mM) and temozolomide (Sigma-Aldrich) (103 mM) were prepared in dimethyl sulfoxide (DMSO) PD0332991 (provided by Pfizer, Peapack, NJ, USA) (10 mM) was prepared in ultrapure water. InhibitorSelect? 384-well protein kinase inhibitor library I The InhibitorSelect protein kinase inhibitor library I (Merck Millipore) was supplied with 160 protein kinase inhibitors in a 384-well plate at a volume of 25 l and a concentration of 10 mM in DMSO and were stored at ?80C. Stock solutions (1 mM) were prepared by dilution in DMSO, and stored at ?20C. Initial screening of the 160 protein kinase inhibitors was performed at 1 M concentration on the Sk-Mel-2 and Sk-Mel-28 cell lines. Cells/well (1103) were seeded in 96-well plates. Plates were incubated overnight at 37C followed by addition of drugs at the appropriate concentrations and incubated for a further 5 days until wells were 80C90% confiuent. At completion of the assay the colorimetric acid phosphatase assay was used to determine cell viability. Proliferation assays and acid phosphatase assay All cells lines SNX-2112 were seeded at 1103 cells/well in 96-well plates except for Malme-3M and WM-115 which were seeded at 2103 cells/well. Plates were incubated overnight at 37C followed by addition of drug at the appropriate concentrations and incubated for a further 5 days until wells were 80C90% confluent. All media were removed and the wells were washed once with phosphate-buffered saline (PBS; Sigma-Aldrich). Paranitrophenol phosphate substrate (7.25 mM; Sigma-Aldrich) in 0.1 M sodium acetate buffer with 0.1% Triton-X (Sigma-Aldrich) pH 5.5 was added to each well and incubated at 37C for 2 h. To stop the reaction 50 l of 1 1 M NaOH was added and the absorbance was go through at 405 nM (reference, 620 nM). Clonogenic assays Malme-3M were seeded at 600 cells/well and Sk-Mel-2 were seeded at 125 cells/well in a 24-well plate. The cells were incubated overnight at 37C. Media were removed and the drugs were added at the appropriate concentrations. Drug made up of medium was removed after 4 days and replaced with drug-free media. The medium was replenished every 4/5 days thereafter. Malme-3M cells were incubated for 17 days and Sk-Mel-2 cells were incubated for 14 days. When ready to stain, media were removed and the cells washed softly with PBS. The plates were then stored at ?20C for 2 h. showed additive anti-proliferative effects in the BRAF mutant cell line Malme-3M. In summary, targeting CDK4 inhibits growth and induces apoptosis in melanoma cells (11) exhibited p16INK4a mutation, promoter methylation or lack of expression occurred in 16, 25 and 82% of melanoma metastases, respectively. The p16INK4a protein binds to CDK4/6 and inhibits conversation with D-type cyclins, which would otherwise stimulate passage through the G1 phase of the cell cycle. The frequent loss of p16INK4a in melanomas suggests that CDK4 activity may be unchecked in melanoma and may play a role in promoting uncontrolled proliferation of melanoma cells. Furthermore, mutation or overexpression of CDK4, combined with amplification of cyclin D1, has been implicated in resistance to BRAF inhibition in V600E-mutated melanoma cells, and amplification of cyclin D1 is usually detected in ~17% of BRAF V600E-mutated human metastatic melanomas (12). The druggable nature of kinases has sparked considerable interest in pursuing CDKs as novel targets in anticancer drug development. Selective inhibition of CDKs may limit the progression of a tumour cell through the cell cycle and facilitate the induction of apoptosis (6,13). Materials and methods Cells and reagents Malme-3M, Sk-Mel-2, Sk-Mel-5, Sk-Mel-28, M14 and Lox-IMVI melanoma cell lines were obtained from the Department of Developmental Therapeutics, National Malignancy Institute (Bethesda, MD, USA). WM-115 and WM-266-4 melanoma cell lines were obtained from the European Association Culture Collection (UK). Malme-3M, Sk-Mel-2, Sk-Mel-5, Sk-Mel-28, M14 and Lox-IMVI cell lines were maintained at 37C with 5% CO2 in RPMI-1640 medium (Sigma-Aldrich, Co. Wicklow, Ireland) with 10% fetal calf serum (FCS; Lonza, Tewkesbury, UK). WM-115 and WM-266-4 were maintained at 37C with 5% CO2 in minimal essential medium (MEM; Sigma-Aldrich) with 10% FCS (BioWhittaker, Walkersville, MD, USA), 2 mM L-glutamine (Life Technologies, Dublin, Ireland), 1 mM non-essential amino acids (Life Technologies) and 1 mM sodium pyruvate (Life Technologies). Stock solutions of fascaplysin (Merck Millipore, Watford, UK) (10 mM), PLX4032 (Sequoia Research Products Ltd., Pangbourne, UK) (10 mM), elacridar (Sigma-Aldrich) (10 mM) and temozolomide (Sigma-Aldrich) (103 mM) were prepared in dimethyl sulfoxide (DMSO) PD0332991 (provided by Pfizer, Peapack, NJ, USA) (10 mM) was prepared in ultrapure water. InhibitorSelect? 384-well protein kinase inhibitor library I The InhibitorSelect protein kinase inhibitor library I (Merck Millipore) was supplied with 160 protein kinase inhibitors in a 384-well plate at a volume of 25 l and a concentration of 10 mM in DMSO and were stored at ?80C. Stock solutions (1 mM) were prepared by dilution in DMSO, and stored at ?20C. Initial screening of the 160 protein kinase inhibitors was performed at 1 M concentration on the Sk-Mel-2 and Sk-Mel-28 cell lines. Cells/well (1103) were seeded in 96-well plates. Plates were incubated overnight at 37C followed by addition of drugs at the appropriate concentrations and incubated for a further 5 days until wells were 80C90% confiuent. At completion of the assay the colorimetric acid phosphatase assay was used to determine cell viability. Proliferation assays and acid phosphatase assay All cells lines were seeded at 1103 cells/well in 96-well plates except for Malme-3M and WM-115 which were seeded at 2103 cells/well. Plates were incubated overnight at 37C followed by addition of drug at the appropriate concentrations and incubated for a further 5 days until wells were 80C90% confluent. All media were removed and the wells were washed once with phosphate-buffered saline (PBS; Sigma-Aldrich). Paranitrophenol phosphate substrate (7.25 mM; Sigma-Aldrich) in 0.1 M sodium acetate buffer with 0.1% Triton-X (Sigma-Aldrich) pH 5.5 was added to each well and incubated at 37C for 2 h. To stop the reaction 50 l of 1 1 M NaOH was added and the absorbance was read at 405 nM (reference, 620 nM). Clonogenic assays Malme-3M were seeded at 600 cells/well.