Fig. 3. (A) Levels of ATF3 mRNA increase on suspension but decline with or without re-adhesion. Semi-quantitative reverse-transcription PCR was used to evaluate levels of ATF3 mRNA in adherent quiescent HKs (Q), Quiescent HKs were suspended with trypsin EDTA or re-adhered to laminin 5 for the indicated times (2, 9, 24 and 48 hours). After 2 hours of suspension or re-adhesion, levels of ATF3 mRNA increased but subsequently declined over time. In controls, levels of GAPDH remained constant. (B) ATF3 protein is upregulated in suspended HKs but declines upon re-adhesion. Confluent quiescent HKs were suspended with trypsin-EDTA and re-adhered onto laminin 5 or held in suspension. Extracts were collected from the quiescent parent population (labeled Quiescent) and from suspended and re-adherent HKs 1-6 hours post suspension. Extracts were immunoblotted with an anti-ATF3 antibody. ATF3 protein was not detectable in quiescent cells, but was elevated in suspended cells throughout the assay (hours 1-6). Levels of ATF3 protein in adherent cells were maximal at 4 hours post activation, after which they declined. (C) Re-adhesion of MKs on laminin 5 suppresses ATF3 protein expression. Laminin 5 null MKs were grown to confluence, suspended and re-plated onto a non-adhesive BSA coated surface or onto a laminin 5-coated surface. Extracts were collected from the quiescent cell population, and from cells plated onto BSA or Laminin 5 at 2 hour intervals from 0-10 hours. Levels of ATF3 in the extracts were examined by immunoblotting. MKs plated onto BSA failed to adhere, upregulated ATF3 protein expression and maintained elevated ATF3 expression for the duration of the assay. MKs plated onto laminin 5 adhered, upregulated ATF3 initially but then suppressed ATF3 expression to baseline by 6 hours. The time course for the downregulation of ATF3 protein was confirmed in at least seven different blot experiments.