Figure 6.

APF-mediated changes in mRNA expression are dependent on palmitoylation of CKAP4 by DHHC2 in NB cells. Primary NB epithelial cells were electroporated with DHHC2 double-stranded siRNA or with nonsense siRNA on day 1 and serum-starved on day 2, and 2.5 nM APF or control peptide was added to the medium on day 3; cells were then cultured for an additional 48 h under conditions of serum starvation. Expression of ZO-1, vimentin, and E-cadherin mRNA was assessed by qRT-PCR as described in Materials and Methods. (A and B) APF alone or in the presence of nonsense siRNA reduced ZO-1 and vimentin mRNA levels by ∼93 and ∼97%, respectively. DHHC2 knockdown blocked this APF-stimulated reduction in ZO-1 and vimentin mRNA levels. (C) APF alone or in the presence of nonsense siRNA dramatically increased E-cadherin mRNA levels, an effect that was also blocked by DHHC2 knockdown. Please note: In contrast to the graphs in A and B, a logarithmic scale was required in C to demonstrate the full range of the increase in E-cadherin expression in response to APF as well as the degree to which this change is blocked by DHHC2 knockdown. ZO-1, vimentin, and E-cadherin mRNA levels were measured in triplicate runs and quantified by normalization to mRNA levels for β-actin using real-time PCR analysis software. The error in the normalized, relative abundance of each mRNA species was propagated forward from the SD of the mean Ct value for each of the experimental samples and the actin control.