FIG 3.

HCMV miR-US5-2 and downregulation of GAB1 affect proliferation of human fibroblasts. (A) NHDF were transfected with negative-control miRNA, miR-US5-2 mimic, or a GAB1 siRNA. 48 h later, cells were trypsinized, counted, and replated at a density of 5,000 cells/well in a 24-well plate in complete media (+FBS), in media lacking FBS (-FBS), or in media lacking FBS but with the inclusion of exogenous EGF (5 ng/ml) (-FBS +EGF). After an additional 48, 96, or 144 h, total viable cells were counted (n = 2). *, P < 0.05 (as determined by two-tailed two-sample t test). (B) At 48 h posttransfection, 2 × 10⁵ cells from the experiment performed as described for panel A were lysed and subjected to immunoblotting for GAB1 and GAPDH. Band intensity was calculated using ImageJ software. The ratio of GAB1 to GAPDH band intensities was set to 1 for the Neg sample, and each subsequent sample ratio is presented as a multiplier of the Neg time point.