Figure 8.

Depletion of Myo9a in Caco-2 cells alters cell morphology, differentiation, Rho-signaling and junctional signaling. (A) Caco-2 cells were transfected with control and Myo9a-targeting siRNAs. After 72 h, the cells were lysed, and expression of Myo9a was analyzed by immunoblotting total cell extacts. α-Tubulin was used as a loading control. (B) Samples of cells treated either with control or Myo9a-targeting siRNAs were analyzed by immunoblotting for the amount of phosphorylated (p-MYPT) and total myosin light chain phosphatase (MYPT). α-Tubulin served as a loading control. (C) Indirect immunofluorescence staining for Myo9a in cells treated with control or Myo9a-targeting siRNAs. Note the disappearance of the junctional staining in cells treated with Myo9a-targeting siRNA. Epifluorescence images; scale bar, 10 μm. (D) The junctional staining of Myo9a does not discriminate between tight and adherence junctions. Confocal sections were taken at the level of the junctions. Caco-2 cells were simultaneously stained for Myo9a, β-catenin, and occludin. Individual stainings and an overlay of the three stainings are shown. Scale bar, 10 μm. (E) Caco-2 cells were transfected with siRNAs as in A and were then fixed and processed for immunofluorescence. Shown are images for the apical marker DPPIV, the basolateral marker NaK-ATPase as well as a set of tight and adherens junction proteins. (F) Transcriptional activity of a reporter plasmid with SRE-dependent luciferase expression. Values obtained with nontargeting siRNAs were set to 100%. (G) Transcriptional activity of NF-κB as measured with a reporter plasmid harboring functional NF-κB-binding sites. Activities were determined in the absence and presence of the Rho-inhibitor C3 transferase (C3). (H and I) Transcriptional activities of β-catenin/TCF and ZONAB, respectively, were measured using luciferase reporter assays. For β-catenin/TCF, results obtained with a reporter plasmid with functional TCF-binding sites as well as with one lacking such sites are shown. For ZONAB, plasmids harboring a promoter with a ZONAB-binding site driving firefly luciferase expression and a promoter with an inactivated binding site but otherwise identical sequence driving renilla luciferase were cotransfected, and the resulting ratios were used to calculate ZONAB activity. Values obtained with nontargeting siRNAs were set to 100%. Values are means ± SD derived from three experiments performed in triplicates.