9. Transfer 1.5 Â 10
6 cells per microtube (prepare as many microtubes as possible) and centrifuge at 9660 Â g for 5 min.
10. Discard the supernatant.
11. Flash freeze pellets in liquid nitrogen and store at À80
C (it is
possible to keep pellets prepared as such for months).
3.2 Methods
for mRNA and Protein
Quantification
By performing Western Blot and RT-qPCR assays on EMT-related
genes and proteins, the mRNA and protein expression levels will
define the physiological state of the cell at the chosen time-points
post TGF-β treatment. Here is a non-exhaustive list of proteins
known as EMT markers: E-cadherin and occludin (epithelial phenotype), N-cadherin and vimentin (mesenchymal phenotype),
snail1, ZEB1, and twist (Transcription factors). In addition, we
also perform every assay on reference genes and proteins (such as
RPLP0, H3, or GAPDH), whose expression levels remain invariant
upon TGF-β treatment in MCF10A cells.
Western Blot and RT-qPCR procedures are standard excepting
cell harvesting.
3.2.1 Western Blot
Cast the gel by following steps 1–11. Before starting, make sure
that your material is clean and dry, and has been previously rinsed
with demineralized water or ethanol.
1. Prepare the casting material as recommended by the supplier.
2. Insert comb into the empty cassette and draw a mark at
0.5–1 cm (see Note 24) below the bottom of the comb teeth:
this will be the level to which the resolving gel will be poured.
3. Remove comb.
4. Use containers that can be closed (Falcon tubes for example),
prepare the resolving and stacking mix, without adding the
APS and TEMED.
5. Add the appropriate volumes of APS and TEMED to the
resolving gel mix, swirl the solution gently but thoroughly
and cast gel. Do not exceed the mark indicating the space left
for the staking gel, and overlay with isopropanol 100% (see
Note 25).
6. Close the container used for the resolving mix and keep on the
side (see Note 26).
7. Allow the gel to polymerize (see Note 27), and just before
adding the stacking gel, return the gel and absorb isopropanol
on a paper towel.
8. Add the appropriate volumes of APS and TEMED in the
stacking gel mix, swirl the solution gently but thoroughly and
distribute over the resolving gel up to the top of the cassette.
EMT Studied by Molecular Biology and DNA-FISH in Human Cells
363
6 cells per microtube (prepare as many microtubes as possible) and centrifuge at 9660 Â g for 5 min.
10. Discard the supernatant.
11. Flash freeze pellets in liquid nitrogen and store at À80
C (it is
possible to keep pellets prepared as such for months).
3.2 Methods
for mRNA and Protein
Quantification
By performing Western Blot and RT-qPCR assays on EMT-related
genes and proteins, the mRNA and protein expression levels will
define the physiological state of the cell at the chosen time-points
post TGF-β treatment. Here is a non-exhaustive list of proteins
known as EMT markers: E-cadherin and occludin (epithelial phenotype), N-cadherin and vimentin (mesenchymal phenotype),
snail1, ZEB1, and twist (Transcription factors). In addition, we
also perform every assay on reference genes and proteins (such as
RPLP0, H3, or GAPDH), whose expression levels remain invariant
upon TGF-β treatment in MCF10A cells.
Western Blot and RT-qPCR procedures are standard excepting
cell harvesting.
3.2.1 Western Blot
Cast the gel by following steps 1–11. Before starting, make sure
that your material is clean and dry, and has been previously rinsed
with demineralized water or ethanol.
1. Prepare the casting material as recommended by the supplier.
2. Insert comb into the empty cassette and draw a mark at
0.5–1 cm (see Note 24) below the bottom of the comb teeth:
this will be the level to which the resolving gel will be poured.
3. Remove comb.
4. Use containers that can be closed (Falcon tubes for example),
prepare the resolving and stacking mix, without adding the
APS and TEMED.
5. Add the appropriate volumes of APS and TEMED to the
resolving gel mix, swirl the solution gently but thoroughly
and cast gel. Do not exceed the mark indicating the space left
for the staking gel, and overlay with isopropanol 100% (see
Note 25).
6. Close the container used for the resolving mix and keep on the
side (see Note 26).
7. Allow the gel to polymerize (see Note 27), and just before
adding the stacking gel, return the gel and absorb isopropanol
on a paper towel.
8. Add the appropriate volumes of APS and TEMED in the
stacking gel mix, swirl the solution gently but thoroughly and
distribute over the resolving gel up to the top of the cassette.
EMT Studied by Molecular Biology and DNA-FISH in Human Cells
363