Journal: Journal of Cellular and Molecular Medicine

Article Title: E2F-1- and E2Ftr-mediated apoptosis: the role of DREAM and HRK

doi: 10.1111/j.1582-4934.2011.01338.x

Figure Lengend Snippet: Overexpression of wtE2F-1 and E2Ftr leads to increased Hrk expression and apoptosis in melanoma cells. (A) Schematic representation of wtE2F-1 and E2Ftr domain structure. Two melanoma cell lines (SK-MEL-2 and A375) were infected with Ad-LacZ (control vector), Ad-wtE2F-1 or Ad-E2Ftr at MOI 100. After 24 hrs of infection, real-time RT-PCR (B) and Western blot analysis (C) were performed as described in Materials and methods. (B) RT-PCR results are expressed as Hrk fold increase relative to control vector-infected cells adjusted for α-actin as an internal control. Each experiment is a representation of three independent experiments performed in duplicate (bars: mean ± S.D.; * P < 0.05 compared with control; n = 3). (C) Cell lysates were subjected to Western blot analysis using HRK, wtE2F-1 or E2Ftr and α-actin antibody. (D) After 48 hrs of infection, the percentage of apoptotic cells showing typical apoptotic nuclei by Hoechst 33258 staining was counted under a fluorescence microscope. The percentage of apoptotic cells (annexin V-PE + cells) were also determined by flow cytometry analysis (E) after 48 hrs of infection, as described in Materials and methods. (F) After 48 hrs of designated infection, the OD405 values of the cell lysates were measured as caspase-9 activity. The results were expressed as the fold change in treated cells over that of the control cells. Values represent mean ± S.D. of three independent experiments (bars: mean ± S.D.; ** P < 0.01 compared with control; n = 3).

Article Snippet: Equal loading of proteins was verified by probing the membrane again with an anti-α-actin primary antibody (1:5000; Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Over Expression, Expressing, Infection, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Reverse Transcription Polymerase Chain Reaction, Staining, Fluorescence, Microscopy, Flow Cytometry, Activity Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: E2F-1- and E2Ftr-mediated apoptosis: the role of DREAM and HRK

doi: 10.1111/j.1582-4934.2011.01338.x

Figure Lengend Snippet: Hrk knockdown by siRNA was associated with significantly reduced wtE2F-1- or E2Ftr-induced apoptosis. SK-MEL-2 and A375 cells were infected with control vector, Ad-wtE2F-1 or Ad-E2Ftr at MOI 100, followed by transfection with control siRNA or Hrk siRNA as indicated. (A) RT-PCR was performed after 24 hrs of transfection as described in Materials and methods. The bar graph is expressed as Hrk fold increase relative to control vector-infected cells adjusted for α-actin as an internal control. Each experiment is a representation of three independent experiments performed in duplicate (Ctrl: control; bars: mean ± S.D.; * P < 0.05; ** P < 0.01 compared with either wtE2F-1 or E2Ftr infection plus control siRNA transfection; n = 3). After 48 hrs of transfection, the percentage of cells showing typical apoptotic nuclei was counted by Hoechst 33258 staining under a fluorescence microscope (B) and determined by annexin V-PE staining by flow cytometry analysis (C). (D) After 48 hrs of transfection, caspase-9 activity was determined as in Figure 1F. Values represent mean ± S.D. of three independent experiments. (Ctrl: control; bars: mean ± S.D.; * P < 0.05; ** P < 0.01 compared with either wtE2F-1 or E2Ftr infection plus control siRNA transfection; n = 3).

Article Snippet: Equal loading of proteins was verified by probing the membrane again with an anti-α-actin primary antibody (1:5000; Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Infection, Plasmid Preparation, Transfection, Reverse Transcription Polymerase Chain Reaction, Staining, Fluorescence, Microscopy, Flow Cytometry, Activity Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: E2F-1- and E2Ftr-mediated apoptosis: the role of DREAM and HRK

doi: 10.1111/j.1582-4934.2011.01338.x

Figure Lengend Snippet: wtE2F-1 and E2Ftr expression resulted in reduced binding of DREAM to the Hrk gene and was correlated with increased HRK up-regulation and apoptosis. SK-MEL-2 and A375 cells were infected as indicated. After 24 hrs of infection, nuclear and cytoplasma protein were extracted. (A) Nuclear proteins (4 μg) were subjected to EMSA using biotin-labelled DRE-Hrk. PCNA Western blot analysis was used as loading control of nuclear proteins from each sample (Ctrl: control virus infected cell nuclear protein lysate; arrow: supershifted band; arrowhead: specific DREAM-Hrk binding). (B) 50 μg of cytoplasma proteins were subjected to Western blot analysis using HRK, wtE2F-1 or E2Ftr, and α-actin antibody. (C) Supershift assay using 10 μg of control virus-infected cell nuclear protein lysate is shown. The specific binding band of DREAM-Hrk (arrowhead) was shifted upward by the addition of anti-DREAM antibody (arrow), but not by control IgG.

Article Snippet: Equal loading of proteins was verified by probing the membrane again with an anti-α-actin primary antibody (1:5000; Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Expressing, Binding Assay, Infection, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: E2F-1- and E2Ftr-mediated apoptosis: the role of DREAM and HRK

doi: 10.1111/j.1582-4934.2011.01338.x

Figure Lengend Snippet: Co-localization of DREAM with wtE2F-1 or E2Ftr in the nuclei and homodimerization of DREAM after wtE2F-1 and E2Ftr overexpression. (A) After 24 hrs of infection in SK-MEL-2 cells, cells were incubated with DREAM antibody followed by Alexa-594 second antibody (shown in red, lower left panel) and E2F-1 antibody followed by Alexa-488 second antibody (shown in green, upper right panel) and then counterstained with DAPI (blue, upper left panel). An overlay (lower right panel) of yellow shows the co-localization of wtE2F-1 or E2Ftr with DREAM in the nuclei (magnification, ×600). Insets represent higher magnifications of the boxed areas (bar = 20 μm). (B) After 24 hrs of infection in SK-MEL-2 and A375 cells, RT-PCR of the DREAM gene was performed as described in Materials and methods. The bar graph is expressed as fold increase relative to control vector-infected cells adjusted for α-actin as an internal control. Each experiment is a representation of three independent experiments performed in duplicate (bars: mean ± S.D.). After 24 hrs of infection in SK-MEL-2 and A375 cells, 60 μg of total proteins from the whole cell lysates (C), cytoplasma (D) and nuclear (E) protein were extracted and subjected to Western blot analysis by using DREAM antibody. α-Actin was used for loading control of the whole cell lysates and cytoplasma fraction. PCNA was used for the loading control of the nuclear fraction (Ctrl: control; arrows: DREAM monomer or homodimer).

Article Snippet: Equal loading of proteins was verified by probing the membrane again with an anti-α-actin primary antibody (1:5000; Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Over Expression, Infection, Incubation, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Western Blot