Journal: BMC Cancer

Article Title: Characterization of ERK Docking Domain Inhibitors that Induce Apoptosis by Targeting Rsk-1 and Caspase-9

doi: 10.1186/1471-2407-11-7

Figure Lengend Snippet: Test compounds induce the intrinsic apoptosis pathway . HeLa ( A ) or TERT ( C ) cells were treated in the absence or presence of test compounds (50 uM) for 5 hours (HeLa) or 16 hours (TERT). Cell lysates were immunoblotted for total and cleaved PARP (arrowhead). Graph on the right shows densitometry quantification of cleaved PARP to α-tubulin ratios and annexinV/PI staining relative control. PARP data represents the mean ± SEM from four independent experiments; annexinV/PI data are representative of two independent experiments. ( B ) HeLa cells were treated with 50 uM of the indicated compound for 5 hours and assayed for cleaved PARP in the presence and absence of the general caspase inhibitor ZVAD-FMK. ( D ) HeLa cells were treated in the absence or presence of 50 uM of the indicated compound for 5 hours and assayed for caspase 8 or 9 activity. Data represent the mean ± SEM from 3 independent experiments. * and # indicates statistical significance compared to untreated controls, p ≤ 0.05 and p ≤ 0.01, respectively. C, Control; E, Etoposide

Article Snippet: The relative protein levels were determined by densitometry scanning (Alpha Innotech), keeping the pixel intensity within the linear range of detection.

Techniques: Staining, Control, Activity Assay

Journal: BMC Cancer

Article Title: Characterization of ERK Docking Domain Inhibitors that Induce Apoptosis by Targeting Rsk-1 and Caspase-9

doi: 10.1186/1471-2407-11-7

Figure Lengend Snippet: Test compounds inhibit ERK-mediated phosphorylation of p90Rsk-1 and Bad . ( A ) HeLa cells were pre-treated for one hour in the presence or absence of 50 uM of the indicated test compounds and then stimulated with EGF (25 ng/ml) for 10 minutes. Immunoblots of phosphorylated p90Rsk-1 (pRsk), total Rsk (Rsk), phosphorylated ERK1/2 (ppERK), and total ERK2 (ERK2). α-tubulin was used as a loading control. Graph shows densitometry quantification of pRsk-1 to total Rsk or ppERK2 to ERK2 ratios. Data represents the mean ± SEM from three independent experiments. ( B ) In vitro kinase assays examining 32 P incorporation into p90Rsk-1 following incubation with active ERK2 and γ- 32 P-ATP for 60 min. in the absence or presence of 1-25 μM of test compounds. Relative phosphate incorporation was quantified by phosphoimager analysis. ( C ) HeLa cells were serum starved overnight and pre-treated for 1 hr with 50 uM indicated test compounds, 10 mM U0126 (U), or 25 mM LY294002 (L) prior to stimulation with or without EGF (25 ng/ml). Immunoblot analysis of Bad phosphorylated on Ser112 (pS112) or Ser136 (pS136) and total Bad. α-tubulin was used as a loading control. Data represents the mean ± SEM from three independent experiments. * and # indicates statistical significance compared to EGF-only treatment (A and C) or untreated controls (B), p ≤ 0.05 and p ≤ 0.01, respectively. C, untreated control; (-) EGF treated control.

Article Snippet: The relative protein levels were determined by densitometry scanning (Alpha Innotech), keeping the pixel intensity within the linear range of detection.

Techniques: Phospho-proteomics, Western Blot, Control, In Vitro, Incubation

Journal: BMC Cancer

Article Title: Characterization of ERK Docking Domain Inhibitors that Induce Apoptosis by Targeting Rsk-1 and Caspase-9

doi: 10.1186/1471-2407-11-7

Figure Lengend Snippet: Test compounds affect the expression of the pro- and anti-apoptotic proteins, Bad and Mcl-1, respectively . Total Bad and Mcl-1 immunoblots from lysates collected from HeLa cells treated for 16 hour with 50 μM of indicated test compounds (top panel) and fold expression quantified by densitometry (bottom panel). α-tubulin was used as a loading control. Data represent the mean ± SEM of three independent experiments. # indicates statistical significance compared to untreated controls, p ≤ 0.01. C, Control

Article Snippet: The relative protein levels were determined by densitometry scanning (Alpha Innotech), keeping the pixel intensity within the linear range of detection.

Techniques: Expressing, Western Blot, Control

Journal: BMC Cancer

Article Title: Characterization of ERK Docking Domain Inhibitors that Induce Apoptosis by Targeting Rsk-1 and Caspase-9

doi: 10.1186/1471-2407-11-7

Figure Lengend Snippet: Test compounds inhibit ERK-mediated phosphorylation of caspase-9 in cells and in in vitro kinase assays . ( A ) HeLa cells transfected with caspase-9 (C287A) were pre-treated for 1 hour with 50 uM of indicated test compound, stimulated with EGF (25 ng/ml), and immunoblotted for phosphorylated caspase-9 (pT125) or total caspase-9. Graph shows the mean ± SEM for relative phosphorylation as determined by densitometry scanning from 3 independent experiments. ( B ) In vitro kinase assays examining 32 P incorporation into caspase-9 following incubation with active ERK2 and γ- 32 P-ATP for 60 min. in the absence or presence of 1-25 μM of the indicated test compound. Relative phosphate incorporation was quantified by phosphoimager analysis. Data show the mean ± SEM of three independent experiments. * and # indicates statistical significance compared to EGF-only treatment ( A ) or untreated controls ( B ), p ≤ 0.05 and p ≤ 0.01, respectively. C, untreated control; (-), EGF treated control; U, U0126

Article Snippet: The relative protein levels were determined by densitometry scanning (Alpha Innotech), keeping the pixel intensity within the linear range of detection.

Techniques: Phospho-proteomics, In Vitro, Transfection, Incubation, Control