Fibronectin levels in response to 4MU treatment.

<p>(A) Fibronectin mRNA levels by qRT-PCR in TM cells treated with 0, 0.5, 1 and 2 mM 4MU for 36 hours. mRNA levels were normalized to 18S RNA levels and presented as a percentage of the control. Error bars are the s.e.m.; n = 3; * P<0.01 by one-way ANOVA. (B) Fibronectin ELISA assay to measure total fibronectin (RIPA lysates and media) protein levels in TM cells. Cells were treated for 4 days with 1 mM 4MU or vehicle-control. Error bars are the s.e.m.; n = 3. * P = 0.0001 by an unpaired Student’s t-test. (C) Western immunoblots of fibronectin protein in conditioned media from TM cells treated with vehicle-control (ctrl) or 1 mM 4MU for 3 and 4 days. The MW markers and 3 day samples are from adjacent lanes of one immunoblot and the 4 days lanes are from adjacent lanes of a different immunoblot. (D) Densitometry of fibronectin showed a significant reduction with 4MU treatment. Fibronectin levels were normalized to total protein loaded and are presented as a percentage of the control. Error bars are the s.e.m.; n = 6 for 3 days, n = 5 for 4 days. * p = 0.039 and ** p = 0.02 by one-way ANOVA. (E) Fibronectin immunostaining of TM cells treated with vehicle-control or 1 mM 4MU for 4 days and viewed by confocal microscopy. Nuclei are stained with DAPI (control) or Draq5 (4MU). Scale bars = 20 µm.