Figure 6

Effect of mPARM-1 and hPARM-1 on proliferation and on anchorage-independent growth of NIH/3T3 cells. (a) NIH/3T3 cells were transfected with pEGFP-N1 (GFP), mParm-1-GFP and hParm-1-GFP expression vectors. After 48h post-transfection, 1x10⁴ cells were resuspended in medium containing 2.5%, 5.0% or 10% of bovine serum. Medium was changed every two days. Cells were fixed and stained after 5 days with 0.2% methylene blue and 50% methanol and photographed at 40X magnification. (b) NIH/3T3 cells were either untransfected or transfected with pcDNA3.1A/Myc-His empty vector, mParm-1-pcDNA3.1A or hParm-1-pcDNA3.1A expression vectors. Cells (5.10³) were plated in soft agar as described in «Colony formation in soft agar». After three weeks, cells were observed with an optical microscope (Ernst Leitz, 6MBH Wetzlar) and representative fields were photographed using a numerical camera (Nikon coolpix 4500; original magnification x40). (c) For each image, the number of colonies formed in soft agar was scored using NIH ImageJ software Version 1.42l. (d) Cells were untransfected or transiently transfected with pEGFP-N1 empty vector, hParm-1-GFP, ∆CT-GFP and ∆EC-GFP expression vectors. As a positive control, activated Ras (EJ 6.6) expression vector was used. For b and c, similar results were obtained using either PARM-1 tagged GFP or Myc-His (data not shown). The same experiment was done as for full-length constructs. All results represent the average of three independent experiments. For panels (c) and (d), the number of colonies in transfected cells was compared to untransfected cells and statistical analysis was performed using one-way analysis of variance (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001).