Journal: BMC Cancer

Article Title: A stable explant culture of HER2/neu invasive carcinoma supported by alpha-Smooth Muscle Actin expressing stromal cells to evaluate therapeutic agents

doi: 10.1186/1471-2407-8-119

Figure Lengend Snippet: Evaluation of p-c-Jun (Ser63) levels and subcellular distribution in MAM-1 co-cultures treated with Iressa by A. Immunofluorescence and B. Flow Cytometry . MAM-1 were subcultured on coverslips or in 6-well plates and grown to ~95% confluence and then treated by replacing the conditioned media with fresh media that contained diluent (.001% DMSO, Control) or 1 μM Iressa for 1 hour prior to fixing and evaluation as described in the methods. A. Immunofluorescent photomicrographs were taken with the 100× objective under oil immersion of cells that were double labeled for p-c-Jun (Ser63) in red (TRITC) and HER2/neu in green (FITC) and counterstained with DAPI (blue) to define the nuclei. B. Flow cytometric analysis of control (left) and Iressa-treated (right) MAM-1 cultures. Cells were dual labeled for p-c-Jun (Ser63) and α-SMA. Density plots compare p-c-Jun (Ser63) expression levels in the α-SMA negative and positive subpopulations in control and Iressa-treated MAM-1 cultures. Mean Channel fluorescent values for p-c-Jun (Ser63) are indicated in the respective quadrants. Histogram analyses for p-c-Jun (Ser63) expression in the α-SMA negative subpopulations were generated from dot plots of p-Jun (Ser63)-PE versus Forward Scatter and gating on the respective the S (small) and L (large) fractions. Histograms of these tumor cell subpopulations demonstrate different baseline levels (Left) of p-c-Jun (Ser63) levels and Iressa responsiveness (Right) in small (S) and large (L) cells. Mean channel fluorescent values for p-c-Jun (Ser63) levels are indicated above the peaks. Parallel samples were dual stained for HER2/neu and p-c-Jun (Ser63) to verify these data (not shown).

Article Snippet: Stained cover slips were washed and mounted in Fluorescent mounting media (cat# HT08, Oncogene Science) containing DAPI for nuclear counterstaining.

Techniques: Immunofluorescence, Flow Cytometry, Control, Labeling, Expressing, Generated, Staining

Journal: BMC Cancer

Article Title: A stable explant culture of HER2/neu invasive carcinoma supported by alpha-Smooth Muscle Actin expressing stromal cells to evaluate therapeutic agents

doi: 10.1186/1471-2407-8-119

Figure Lengend Snippet: Effect of Iressa on phosphorylation of p44/42 MAPK by (A) Immunofluorescence and phosphorylation of p44/42 MAPK and MEK1/2 by (B) flow cytometry in MAM-1 co-cultures . MAM-1 were subcultured on coverslips or in 6-well plates and grown to ~95% confluence and then treated by replacing the conditioned media with fresh media that contained diluent (.001% DMSO, Control) or Iressa for 2 hours prior to fixing and evaluation as described in the methods. A. Immunofluorescent photomicrographs were taken with the indicated objectives of cells that were double labeled for phospho-p44/42 MAPK (Thr202/Tyr204) in green (FITC) and α-SMA in red (TRITC) and counterstained with DAPI (blue) to define the nuclei. B. Dose-response for phosphorylation of p44/42 MAPK and MEK1/2 in MAM-1 co-cultures by flow cytometry. MAM-1 were treated as described above and dual-labeled for ErbB-2 and the indicated phospho-specific antigen. Bars represent Mean channel fluorescent values for pp44/42 MAPK (Thr202/Tyr204) (Blue Bars) or pMEK1/2 (Ser217/221) (Red Bars) in ErbB2+ (Solid Bars) and ErbB2-(Shaded Bars) subpopulations.

Article Snippet: Stained cover slips were washed and mounted in Fluorescent mounting media (cat# HT08, Oncogene Science) containing DAPI for nuclear counterstaining.

Techniques: Phospho-proteomics, Immunofluorescence, Flow Cytometry, Control, Labeling

Journal: BMC Cancer

Article Title: A stable explant culture of HER2/neu invasive carcinoma supported by alpha-Smooth Muscle Actin expressing stromal cells to evaluate therapeutic agents

doi: 10.1186/1471-2407-8-119

Figure Lengend Snippet: Effect of Iressa on (A) PCNA activity and (B) Signal transduction in the tumor and stromal cell populations of MAM-1 co-cultures . A. MAM-1 co-cultures were treated for 24 h with fresh media in the absence (Control) or presence of 1 μM Iressa then fixed and stained with PC10-TRITC to detect PCNA reactivity (red) and counterstained with DAPI to identify the nuclei (blue) Photomicrographs were taken with the 100× objective under oil immersion. The PCNA index of control tumor cells is >95% and <12% in the Iressa treated tumor cells. In the stroma adjacent to the Iressa treated tumor nest, PCNA index is >95%. Note the presence of apoptotic cells in DAPI stained tumor cells treated with Iressa (arrow). B. Differential effect of Iressa on PCNA, phospho-p44/42 MAPK (Thr202/Tyr204) and phospho-MEK1/2 (Ser217/221) in the ErbB-2 Negative (Stromal) and ErbB-2 Positive (Tumor) populations in MAM-1 treated for 24 h with 1 μM Iressa. Samples were dual-labeled for ErbB-2 and the indicated antigen and evaluated by dual-color flow cytometry. Bars represent the Mean Channel Fluorescent Values for phospho-p44/42 MAPK (Blue Bars), phospho-MEK1/2 (Red Bars) or PCNA (Black Bars). Following Iressa treatment, the ErbB-2 positive population decreased by 44% and the α-SMA positive population increased 3-fold in these MAM-1 co-cultures.

Article Snippet: Stained cover slips were washed and mounted in Fluorescent mounting media (cat# HT08, Oncogene Science) containing DAPI for nuclear counterstaining.

Techniques: Activity Assay, Transduction, Control, Staining, Labeling, Flow Cytometry