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. 2009 Apr 20;106(18):7461–7466. doi: 10.1073/pnas.0902368106

Fig. 6.

Fig. 6.

Effect of exogenous addition of gangliosides on cell motility, morphology, and expression of epithelial vs. mesenchymal cell markers. NMuMG cells (2 × 105 per well in 6-well plates) were cultured in 10% FBS/DMEM overnight. Cells were preincubated with 50 μM GM2 or Gg4 prepared as described in the Fig. 5 legend for 8 h, then incubated for 48 h in 10% FBS/DMEM containing 1 ng/mL TGF-β plus 50 μM GM2 or Gg4. (A) Cell motility was determined as described in SI Materials and Methods and the Fig. 2 legend. For analysis of cell morphology and expression of EMT markers, NMuMG cells (5 × 104 per well in 12-well plates) were cultured overnight as above. Cells were preincubated with 50 μM GM2 or Gg4 for 18 h in 2% FBS/DMEM, then incubated for 36 h in 10% FBS/DMEM containing 1 ng/mL TGF-β plus 50 μM GM2 or Gg4. Cells were fixed and stained, and photos were taken (B). Expression of marker proteins was analyzed by Western blot. Representative results (C) and relative level of expression normalized with loading control (D) are presented as mean ± SD from triplicate experiments. 1 indicates no treatment; 2, TGF-β treatment alone; 3, GM2 plus TGF-β treatment; and 4, Gg4 plus TGF-β treatment. P > 0.05. *, P = 0.01 to 0.05. **, P = 0.001 to 0.005. ***, P < 0.001.