Figure 6. Serine 4 modulates Snail2-mediated EMT.

Snail2-HA wild type and the Snail2-S4A or the Snail2-S4D mutants were stably transfected into MDCK cells and selected clones analyzed for phenotype and EMT markers. (A) Phenotypic characterization of three representative Snail2-S4A clones (#1, #2 and #5) and two representative Snail2-S4D clones (#2 and #3) compared to Snail2-HA cells (clone #3). Panels a to f, phase contrast images; g to l, immunofluorescence staining for E-cadherin (green). Nuclei are stained with DAPI (blue). Bars, 200 µm. (B) Western blot analysis of the indicated clones for E-cadherin (upper) and vimentin (middle); migration of the indicated proteins is shown in kDa at the left. (C) E-cadherin promoter activity in the indicated MDCK clones. Reporter assays were performed as described in Material and Methods, and relative luciferase units (RLU) normalized to the activity obtained in MDCK-Snail2-HA cells. Results show the mean of duplicate experiments, performed on triplicate samples, +/− s.d. (D) Western blot analysis of the indicated clones for Snail2-HA, wt and mutants, detection using anti-HA antibodies. Migration of Snail2-HA wild type and mutants (32 kDa) is indicated at the left. α-tubulin (55 kDa) was used as loading control in (B) and (D). Similar results were obtained for all isolated clones. (E) The stability of Snail2-HA and the indicated Snail2-HA mutants in HEK293T cells was determined by incubation in the presence of the translational inhibitor cycloheximide for the indicated time periods. Upper, Western blot analysis of Snail2-HA levels of one representative experiment from Snail2-HA wild type and mutants with anti-HA antibodies; α-tubulin was used as a loading control. Bottom, densitometric quantification of the relative amounts of Snail2-HA and the two mutants at the indicated time points. Results show the mean of three independent experiments +/− s.d.