Journal: Cancer research

Article Title: An in vivo reporter to quantitatively and temporally analyze the effects of CDK4/6 inhibitor-based therapies in melanoma

doi: 10.1158/0008-5472.CAN-15-3384

Figure Lengend Snippet: A. Representative annexin V staining of melanoma lines treated with trametinib (5 nM) and palbociclib (0.5 μM) treatment alone or a combination of both compounds for 48 hours (n=3, error bars=SD from triplicate samples, *p<0.05). B. A375 cells were treated with single agent or combination of trametinib and palbociclib for 24 hours. Lysates were analyzed by RPPA. The heatmap shows the most significantly regulated proteins (p<0.01). C. Elevated levels of Bim-EL in trametinib and combo-treated lysates. D. 1205Lu cells were transfected with siRNA to Bim in the presence or absence of trametinib and palbociclib. Knockdown of Bim rescued apoptotic phenotype elicited by trametinib and palbociclib treatments. E. Fold change in BIRC5/survivin regulation after 24 hours of treatment with indicated inhibitors. Two independent sets of tests were carried out and representative data is shown. F. Western blotting for survivin in resistant (CHL-1, Bowes, SKMEL207) and sensitive (A375, WM793, 1205Lu, SBcl2, WM1346, WM1366) cell lines in basal state as well as following treatment with trametinib and/or palbociclib for 48 hours. G. NRAS mutant melanoma tumor explant treated with DMSO, trametinib (50 nM), palbociclib (0.5 μM) or the combination.

Article Snippet: CHL-1 and A375 cells (purchased from ATCC, Manassas, VA in 2013 and 2005 respectively) were cultured in DMEM with 10% FBS.

Techniques: Staining, Transfection, Knockdown, Western Blot, Mutagenesis

Journal: British Journal of Pharmacology

Article Title: Model‐free and kinetic modelling approaches for characterising non‐equilibrium pharmacological pathway activity: Internalisation of cannabinoid CB 1 receptors

doi: 10.1111/bph.14684

Figure Lengend Snippet: CB 1 receptor agonist‐induced internalisation potencies ( p EC 50 ± SEM) in 3HA‐hCB 1 HEK cells from the traditional “snapshot” equilibrium analysis across six stimulation time points ( n = 3, unless indicated otherwise).

Article Snippet: The 3HA‐hCB HEK293 cell line (ATCC, Manassas, VA; Cat# CRL‐1573, RRID:CVCL_0045 transfected with 3HA‐hCB ; Cawston et al., 2013 ) was cultured in DMEM supplemented with 10% FBS and 250‐μg ml ‐1 ZeocinTM, in a 5% CO 2 , 37°C humidified incubator.

Techniques:

Journal: British Journal of Pharmacology

Article Title: Model‐free and kinetic modelling approaches for characterising non‐equilibrium pharmacological pathway activity: Internalisation of cannabinoid CB 1 receptors

doi: 10.1111/bph.14684

Figure Lengend Snippet: The potency/efficacy of CB 1 receptor agonists for internalisation in 3HA‐hCB 1 HEK cells using the model‐free approach with the inverse of mean residence time as the response metric

Article Snippet: The 3HA‐hCB HEK293 cell line (ATCC, Manassas, VA; Cat# CRL‐1573, RRID:CVCL_0045 transfected with 3HA‐hCB ; Cawston et al., 2013 ) was cultured in DMEM supplemented with 10% FBS and 250‐μg ml ‐1 ZeocinTM, in a 5% CO 2 , 37°C humidified incubator.

Techniques:

Journal: British Journal of Pharmacology

Article Title: Model‐free and kinetic modelling approaches for characterising non‐equilibrium pharmacological pathway activity: Internalisation of cannabinoid CB 1 receptors

doi: 10.1111/bph.14684

Figure Lengend Snippet: Summary of parameter estimates from the quasi‐steady state internalisation model for agonist‐induced internalisation in 3HA‐hCB 1 HEK cells on stimulation with six CB 1 receptor agonists

Article Snippet: The 3HA‐hCB HEK293 cell line (ATCC, Manassas, VA; Cat# CRL‐1573, RRID:CVCL_0045 transfected with 3HA‐hCB ; Cawston et al., 2013 ) was cultured in DMEM supplemented with 10% FBS and 250‐μg ml ‐1 ZeocinTM, in a 5% CO 2 , 37°C humidified incubator.

Techniques:

Journal: British Journal of Pharmacology

Article Title: Model‐free and kinetic modelling approaches for characterising non‐equilibrium pharmacological pathway activity: Internalisation of cannabinoid CB 1 receptors

doi: 10.1111/bph.14684

Figure Lengend Snippet: Concentration–response curves generated by model‐free analysis of kinetic internalisation data (the inverse of mean residence time) showing 3HA‐hCB1 HEK cells signalling on stimulation with a panel of six agonists: CP55,940, WIN55,212‐2, AEA, 2‐AG THC and BAY59,3074 Symbols represent model‐free metric calculated from raw data (demonstrating mean ± SEM of technical duplicates) and the curves shown are classic three‐parameter Emax model fits (n H constrained to one)

Article Snippet: The 3HA‐hCB HEK293 cell line (ATCC, Manassas, VA; Cat# CRL‐1573, RRID:CVCL_0045 transfected with 3HA‐hCB ; Cawston et al., 2013 ) was cultured in DMEM supplemented with 10% FBS and 250‐μg ml ‐1 ZeocinTM, in a 5% CO 2 , 37°C humidified incubator.

Techniques: Concentration Assay, Generated

Journal: British Journal of Pharmacology

Article Title: Model‐free and kinetic modelling approaches for characterising non‐equilibrium pharmacological pathway activity: Internalisation of cannabinoid CB 1 receptors

doi: 10.1111/bph.14684

Figure Lengend Snippet: The fitting results of the quasi‐steady state internalisation model for agonist‐induced internalisation in 3HA‐hCB1 HEK cells on stimulation with six CB1 receptor ligands: CP55,940 (a), WIN55,212‐2 (b), AEA (c), 2‐AG (d), THC (e), and BAY59,3074 (f). The line here represents the prediction from quasi‐steady state model of agonist‐induced internalisation for “live‐at‐start” assay and the symbols (mean ± SEM) here are the observed data from the internalisation assay

Article Snippet: The 3HA‐hCB HEK293 cell line (ATCC, Manassas, VA; Cat# CRL‐1573, RRID:CVCL_0045 transfected with 3HA‐hCB ; Cawston et al., 2013 ) was cultured in DMEM supplemented with 10% FBS and 250‐μg ml ‐1 ZeocinTM, in a 5% CO 2 , 37°C humidified incubator.

Techniques: Internalisation Assay

Journal: British Journal of Pharmacology

Article Title: Model‐free and kinetic modelling approaches for characterising non‐equilibrium pharmacological pathway activity: Internalisation of cannabinoid CB 1 receptors

doi: 10.1111/bph.14684

Figure Lengend Snippet: Comparison of the potency/efficacy of CB1 receptor agonists for internalisation in 3HA‐hCB1 HEK cells derived from model‐free method (the inverse of mean residence time) and kinetic modelling approach (the quasi‐steady state internalisation model). (a) Correlation between potencies of CB1 receptor ligands for internalisation derived from kinetic modelling (pKSS) and model‐free approach (pEC50; r2 = 0.87). (b) Correlation between efficacies of CB1 receptor ligands for internalisation derived from kinetic modelling (logkint) and model‐free approach (logEmax; r2 = 0.98). Here, the blue dashed line indicates the trend line from linear regression

Article Snippet: The 3HA‐hCB HEK293 cell line (ATCC, Manassas, VA; Cat# CRL‐1573, RRID:CVCL_0045 transfected with 3HA‐hCB ; Cawston et al., 2013 ) was cultured in DMEM supplemented with 10% FBS and 250‐μg ml ‐1 ZeocinTM, in a 5% CO 2 , 37°C humidified incubator.

Techniques: Comparison, Derivative Assay

Journal: British Journal of Pharmacology

Article Title: Model‐free and kinetic modelling approaches for characterising non‐equilibrium pharmacological pathway activity: Internalisation of cannabinoid CB 1 receptors

doi: 10.1111/bph.14684

Figure Lengend Snippet: Representative “live‐at‐start” internalisation time courses, showing agonist‐induced internalisation of surface 3HA‐hCB1 in HEK cells upon stimulation with concentration series of CP55,940 (a), WIN55,212‐2 (b), AEA (c), 2‐AG (d), THC (e), and BAY59,3074 (f). Symbols represent raw data (demonstrating mean ± SEM of technical duplicate) and the lines represent the prediction from one phase decay model with parameter “Plateau” constrained to zero

Article Snippet: The 3HA‐hCB HEK293 cell line (ATCC, Manassas, VA; Cat# CRL‐1573, RRID:CVCL_0045 transfected with 3HA‐hCB ; Cawston et al., 2013 ) was cultured in DMEM supplemented with 10% FBS and 250‐μg ml ‐1 ZeocinTM, in a 5% CO 2 , 37°C humidified incubator.

Techniques: Concentration Assay

Journal: British Journal of Pharmacology

Article Title: Model‐free and kinetic modelling approaches for characterising non‐equilibrium pharmacological pathway activity: Internalisation of cannabinoid CB 1 receptors

doi: 10.1111/bph.14684

Figure Lengend Snippet: Representative “live‐at‐start” internalisation concentration–response curves, showing agonist‐induced internalisation of surface 3HA‐hCB1 in HEK cells upon stimulation for six different time points with CP55,940 (a), WIN55,212‐2 (b), AEA (c), 2‐AG (d), THC (e), and BAY59,3074 (f). Symbols represent raw data (demonstrating mean ± SEM of technical duplicate) and the curves shown are classic three‐parameter Emax model fits (n H constrained to one)

Article Snippet: The 3HA‐hCB HEK293 cell line (ATCC, Manassas, VA; Cat# CRL‐1573, RRID:CVCL_0045 transfected with 3HA‐hCB ; Cawston et al., 2013 ) was cultured in DMEM supplemented with 10% FBS and 250‐μg ml ‐1 ZeocinTM, in a 5% CO 2 , 37°C humidified incubator.

Techniques: Concentration Assay