Figure 5.

SUMOylation is necessary for TGFβ-induced p27 nuclear accumulation. (A) Western blot analysis of endogenous p27 expression level in MCF7 cells in the presence or absence of HA-tagged dominant-negative Ubc9 vector (HA-tagged Ubc9^DN), and treated or not with TGFβ (20 ng/ml) for 3 h. Vinculin was used as loading control and phosphorylated Smad2 (pSer 465/467) was used as a marker of TGFβ signaling activation. Normalized p27 level (p27/vinculin ratio) relative to the level in empty vector-transfected, untreated cells was determined by densitometric analysis of the blots (±SD) (right graph). (B) Immunofluorescence analysis of p27 (green), SUMO1 (red), and Ubc9^WT or Ubc9^DN (blue) in MCF7 cells transiently transfected with HA-tagged Ubc9^WT or Ubc9^DN, and treated or not with TGFβ (20 ng/ml) for 3 h. Ubc9-expressing cells were identified using an anti-HA antibody (blue). Red arrows indicate cells expressing Ubc9^WT with p27 nuclear accumulation. Green arrow indicates a cell expressing Ubc9^DN without p27 nuclear accumulation. Nuclear p27 fluorescence intensity in at least 20 cells expressing or not Ubc9^WT or Ubc9^DN and treated or not with TGFβ was calculated using the Volocity™ Software (right graph). Significance was calculated using the Mann–Whitney unpaired t-test. (C) Immunofluorescence analysis of EGFP-p27 expression and localization (green) in HeLa cells transiently transfected with HA-SUMO1, untagged Ubc9, and EGFP-p27^WT or EGFP-p27^K134R mutant, and then treated with TGFβ (20 ng/ml) for 3 h. Nuclear staining is reported in blue. The percentage of cells expressing nuclear (%N) or nuclear + cytoplasmic (%N/C) EGFP-p27^WT or EGFP-p27^K134R before and after 30 min, 1 h, and 3 h of TGFβ treatment was determined (right table). Data were collected by using a 40× objective and scoring all transfected cells in 10 randomly selected fields per each condition.