Fig. 3

FBLN5 induces cell death in transformed COS-7 cells but not in normal CV-1 cells. a, b Clonogenicity assay upon stable overexpression of FBLN5 in CV-1 and COS-7 cells. FBLN5, tBID (positive control) and pcDNA 3.1 empty vector (negative control) were transfected into CV-1 and COS-7 cells, selected and maintained in 2.5 mg/mL and 1.0 mg/mL G418 containing media, respectively. When there were no cells left in the untransfected (untreated) population, plates were fixed with 4% PFA, and colonies stained with crystal violet and counted. Histogram (b) represents average ± SD number of colonies in the representative image (a) with two independent transfections. c, d cell death in CV-1 and COS-7 cells upon FBLN5 transfection as determined by phenotypic inspection. CV-1 and COS-7 cell were co-transfected with GFP and either FBLN5, luciferase (luc, negative control) or tBID (positive control) in 1:4 ratio (1 GFP: 4 test plasmid) and 48 h post-transfection, fluorescent green cells were imaged (c) and manually counted (~ 3000 COS-7 cells and ~ 2500 CV-1 cells; (d)). Rounded, clustered and/or cells with disfigured morphology were considered dead. No green cells could be detected in CV-1 cells transfected with tBID and 100% was added manually in the chart. c indicates a representative image from more than 75 fields of view. Histogram (d) represents average ± SD from three independent transfections. Statistical significance was calculated using two-tailed Student’s t-test (**p ≤ 0.005, ***p ≤ 0.0005)