TGF-beta is a main driver of epithelial-to-mesenchymal transition, in short EMT, which can occur both in normal and in cancer cells. The methods that are described in our protocol allow researchers to measure the impact of modulators on both TGF-beta SMAD signaling and TGF-beta-induced EMT. The untransformed NMuMG cells, which were established from a mouse mammary gland, is a frequently used model system for TGF-beta-induced EMT.
TGF-beta provokes in these cells, these NMuMG cells a very strong EMT response. A similar response can also be seen in breast cancer cells where it contributes to cancer progression. The combined staining of epithelial micro E-cadherin and organization of filamentous-actin helps to validate and better visualize the morphological changes that occur during epithelial-to-mesenchymal transition.
The acquisition of a mesenchymal phenotype by cancer cells has been linked to an increased ability of cells to migrate, to invade, and to metastasize, as well as to chemotherapy resistance. This method may provide important insights into cancer progression, but also into other processes such as tissue fibrosis. While attempting this protocol, keep in mind that cell density should be well-controlled.
High density of the cells makes epithelial-to-mesenchymal transition difficult. With this visual demonstration, researchers will get a good grasp on how to perform TGF-beta signaling and TGF-beta-induced EMT experiments. To perform indirect immunofluorescent staining of E-cadherin, begin by placing sterile 18 millimeter square glass coverslips in six-well plates, one coverslip per well.
Seed 100, 000 NMuMG cells with two milliliters of complete DMEM into each well and allow the cells to adhere overnight. Non-transformed NMuMG cells are frequently used as a model system to investigate TGF-beta-induced EMT. On the next day, gently move the coverslips with adherent cells to a new six-well plate and add two milliliters of culture medium to the wells.
Treat the cells with ligand buffer as a control or five nanograms per milliliter of TGF-beta for two days. The added ligand buffer should be the same volume as the added TGF-beta. After the treatment, remove the culture medium and gently wash the cells twice with one milliliter of pre-warmed PBS.
Fix the cells by adding one milliliter of 4%paraformaldehyde and incubating for 30 minutes at room temperature, then gently wash the cells twice with one milliliter of PBS. Permeabilize the fixed cells with 0.1%Triton X-100 for 10 minutes at room temperature and wash the cells twice with PBS. Block the non-specific protein binding to cells with 5%BSA in PBS for one hour at room temperature, then repeat the washes with PBS.
Add the primary antibody against E-cadherin to the top of each coverslip and incubate for one hour at room temperature. Remove the primary antibody and wash each coverslip with PBS three times. Add the Alexa Fluor 555 secondary antibody and the Alexa Fluor 488 phalloidin to the top of each coverslip.
Then cover with aluminum foil and incubate for one hour at room temperature. Remove the secondary antibody and wash the coverslip three times with PBS. Mount the coverslip with the cells facing downward onto glass slides using mounting medium with DAPI and store the mounted slides in a box at four degrees Celsius protected from light.
Observe the staining with SP8 confocal microscopy. In the MCF10A-Ras cell line, the phosphorylation of SMAD2 significantly increased in response to TGF-beta stimulation, while the expression of total SMAD2 and 3 was not affected. TGF-beta also markedly induced the luciferase reporter in the MCF10A-Ras cell line compared to non-treated cells.
Well-characterized direct transcriptional gene targets of TGF-beta, including SMAD7 and SERPINE1, were highly expressed in TGF-beta-treated MCF10A-Ras breast cells. NMuMG epithelial cells treated with TGF-beta changed from a classic epithelial morphology to a spindle-shaped mesenchymal-like morphology. Consistent with the morphological changes, TGF-beta treatment led to an increase in the protein expression of mesenchymal markers.
In contrast, E-cadherin, an epithelial marker, was down-regulated after two days of TGF-beta treatment. Quantitative real-time polymerase PCR was used to investigate the gene expression of EMT markers after TGF-beta stimulation. CDH1 was significantly decreased while mesenchymal markers were increased.
Upon TGF-beta stimulation, NMuMG cells expressed less E-cadherin than the cells in the control group. Moreover, the cells formed more actin stress fibers in the presence of TGF-beta. Treatment with GW788388 inhibited TGF-beta-induced SMAD2 phosphorylation in a dose-dependent manner.
Additionally, the TGF-beta-mediated phosphorylation of SMAD2 was blocked by SB-431542 treatment. Both SB-431542 and GW788388 significantly inhibited the TGF-beta-induced nuclear translocation and accumulation of SMAD2 and 3 in NMuMG cells. Following this procedure, immunofluorescent staining of other epithelial-to-mesenchymal macroproteins can be performed by changing the specific primary and secondary antibodies to investigate the expression level and the localization of these proteins within the cell.
