Figure 4.

The total proteins from the HCC cells with or without EMT were extracted and applied 2-D DIGE and MALDI-TOF/TOF MS analysis. (A) A 2-D DIGE pattern (Gel 4). According to the experimental design, protein samples from each HCC cultures were extracted and labeled with CyDyes, Cy2-labeled internal standard sample (pool), Cy3-labeled sample (EMT HCC) and Cy5-labeled sample (HCC) were mixed and separated in a 2-D DIGE gel (IEF on pH 4-7 strips followed by 12.5% second dimension SDS-PAGE), the DIGE gel was then visualized with a Typhoon 9400 scanner. Marked protein spots indicate the significantly expressed proteins which corresponding to the proteins listed in Table 3. (B) A typical example is the protein spot 896, whose abundance increased by 4.27 folds after EMT. (C) LC-MS analyzed spot 896 and identified the protein sequence of peptides extracted from spot 896. (D) Western blotting analysis confirmed TG2 expression level in Hep3B, 97L or Huh7 cells after NSF-CM or CAF-CM incubation.