Figure 1. GWL overexpression promotes cell transformation in immortalised mammary gland cells and in primary human fibroblasts.
(A-C) MCF10A cells were stably infected with either empty pMXs vector (CT) or plasmids coding for wild type (WT) or hyperactive (K72M) GWL. A MCF10A stable clone overexpressing the V12RAS oncogene was used as a positive control. Cell proliferation (A), cell—cell contact inhibition (B) and anchorage-independent cell growth (C) were assayed in these cells. Colonies stained by crystal violet in normal seeded plates (cell—cell contact inhibition) or in soft-agar plates (anchorage-independent cell growth) were counted and results presented in a graph. Results are shown as the mean of three different experiments ± SD. Two-tailed unpaired Student t tests were performed to determine statistical relevance; significant p values are shown. Scale bars, 60 μm. (D) Primary human fibroblasts were infected with empty pMXs vector (CT) or with plasmids coding for the indicated GWL proteins. FACS profile and β-Galactosidase staining (blue) of these cells are shown. (E,F) Human fibroblasts expressing hTERT and SV40 T large antigen were infected with an empty vector (CT) or with plasmids encoding WT or K72M GWL. An hTERT-SV40-V12Ras cell line was used as a positive control. Cell proliferation (E) and anchorage-independent cell growth (F) are shown. Results are the mean ± SD of three different experiments. Two-tailed unpaired Student’s t tests were performed to determine the statistical relevance; significant p values are shown. Scale bars, 100 μm. SD, standard deviation.
Figure 1—figure supplement 1. G44S kinase dead form of GWL in MCF10A cell lines could act as dominant-negative of endogenous GWL.
Figure 1—figure supplement 2. NIH3T3 cells were stably infected with either the pMXs empty vector (CT) or the GWL wild type (WT) or hyperactive mutant form (K72M).
