DNA-FISH labeling. For each harvesting day (previously determined), grow three cell samples in parallel: one for the untreated
condition (as a negative control), one for the transient TGF-β
treatment, and one for the continuous TGF-β treatment (see
Note 20).
3.1.2 TGF-β Treatment
1. For transient TGF-β treatment, add 1.25 μL of TGF-β stock
solution (see Note 20) for 10 mL of growing media (for a final
concentration of 2.5 ng/mL) for 4 h, then replace the TGF-β
supplemented growing media with non-supplemented
MCF10A growing media.
2. For continuous TGF-β treatment, cultivate cells in 2.5 ng/mL
TGF-β supplemented growing media (and Note 20). Refresh
the TGF-β every 48 h.
3. Regularly observe cells by light microscopy to follow phenotypic changes upon transient or continuous TGF-β treatments
(see Fig. 1a, b and Note 21).
3.1.3 Harvesting Cells
for Western Blot
Harvest cells for analysis at the desired post- TGF-β treatment timepoints (see Note 22).
1. Put the cell culture dish on ice. We consider that a 10 mm cell
culture dish at 80% confluence contains approximately 5 Â 10
6
MCF10A cells.
2. Rinse cells with 5 mL of cold 1Â PBS containing protease
inhibitors.
3. Discard the 1Â PBS containing protease inhibitors.
4. Add 2 mL of cold 1Â PBS containing protease inhibitors.
5. Scratch cells with a cell spatula making sure that the harvested
cells are in the fluids (tilt the cell culture dish).
6. Transfer the harvested cells in a 15 mL tube.
7. Repeat steps 4–6 once.
8. Centrifuge the 15 mL tube at 1250 Â g at 4
C for 15 min.
9. Discard the supernatant.
10. Add 500 μL of lysis buffer.
11. Incubate 30 min at 4
C.
12. Optional: At this stage, proteins concentrations can be quantified. We use the DC protein assay from Bio-Rad.
13. Add 150 μL of 4Â Laemmli sample buffer with
β-mercaptoethanol.
14. Store at À20
C.
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