Journal: PLoS ONE

Article Title: Oncogene Activation Induces Metabolic Transformation Resulting in Insulin-Independence in Human Breast Cancer Cells

doi: 10.1371/journal.pone.0017959

Figure Lengend Snippet: (A) Plasma membrane proteins were isolated from MCF10A cells cultured with insulin (5 µg/ml) and MCF10HER2 cells cultured without insulin. Isolated membrane proteins were probed for GLUT1, GLUT3 and GLUT4. Transferrin receptor was used as a plasma membrane loading control. Amounts of plasma membrane-localized GLUT1 and GLUT3 appeared to be similar in both samples. Although levels of the insulin-responsive transporter GLUT4 were higher in membrane preparations from MCF10A cells cultured in the presence of insulin, GLUT4 was readily detectable at the plasma membrane in MCF10HER2 cells maintained long-term in the absence of insulin in serum-free media. (B) MCF10HER2 cells were maintained in serum-free media with out insulin and MCF10A cells were incubated for 18 hours in the absence of insulin in serum-free media, then both cell lines were cultured with or without insulin for 30 minutes. Plasma membrane localized proteins were harvested and probed for GLUT4. In MCF10HER2 cells cultured without insulin basal levels of plasma membrane localized GLUT4 were calculated to be 44% higher than basal GLUT4 plasma membrane levels in MCF10A cells cultured without insulin. In both cell types, insulin induced an increase in GLUT4 at the plasma membrane. The highest levels were observed in the MCF10HER2 cells plus insulin condition. (C) bands in (B) were quantified (optical density) by Quantity One software from Bio-Rad Laboratories. Levels of GLUT4 were normalized to transferrin receptor levels in each context and expressed as a ratio. Relative normalized expression is compared to MCF10A minus-insulin results.

Article Snippet: IR and IGF-IR immunoprecipitates (50% of eluent), whole cell lysates (100 ug) and isolated cell surface proteins (25 ug) were separated on a 7.5% SDS-polyacrylamide gels and transferred to PVDF membranes by semi-dry electrophoretic transfer with a Bio-Rad Transblot SD unit.

Techniques: Isolation, Cell Culture, Incubation, Software, Expressing