13. Stock solution of human recombinant Tumour Necrosis Factor
alpha (hTNFα) (StemCell Technologies or any equivalent reference from other suppliers) at 50 μg/mL prepared following
providers’ recommendation) and stored at À20
C.
14. Control medium composed of DMEM/F12 supplemented
with 5% horse serum, 10 ng/mL cholera toxin, 10 μg/mL
insulin, 0.5 μg/mL hydrocortisone, 20 ng/mL human recombinant epidermal growth factor, 1% Penicillin/Streptomycin,
1% L-Glutamine (see Note 2).
15. EMT inducing medium (EMT-IM): control medium plus
TGF-β (5 ng/mL), TNF-α (10 ng/mL), and IL-6 (10 ng/
mL) (see Notes 2 and 3).
2.4 Real-Time PCR
1. Total RNA extraction kit (PureLink™ RNA Mini Kit, ThermoFisher) or any equivalent reference from other suppliers.
2. High-Capacity cDNA Reverse Transcription kit (ThermoFisher) or any equivalent reference from other suppliers.
3. FastStart Universal SYBR Green Master mix (Roche).
4. Real-time PCR primers:
SNAIL1: FW 5
0 -GCTGCAGGACTCTAATCCAGA-3
0 ; RV 50 -ATCTCCGGAGGTGGGATG-3
0 [11].
SNAIL2: FW 5
0 -TTCGGACCCACACATTACCT-3
0 ; RV 50 -TCTCCCCCGTGTGAGTTCTA-3
0 .
ZEB1: FW 5
0 - AGGGCACACCAGAAGCCAG-3
0 ; RV 50 -GAGGTAAAGCGTTTATAGCCTCTATCA-3
0 [12].
TBP: FW 5
0 -CACGAACCACGGCACTGATT-3
0 ; RV 50 -TTTTCTTGCTGCCAGTCTGGAC-3
0 [12].
36B4: FW 5
0 -GCTGATGGGCAAGAACACCA-3
0 ; RV 50 -CCGGATATGAGGCAGCAGTT-3
0 [11].
SMA: FW 5
0 -GAGCGTGAGATTGTCCGGGA-3
0 ; RV 5
0 -
AAGGGAGGATGAGGATGCGG-3
0 .
5. Real-time PCR thermocycler.
2.5 Flow Cytometry
1. Flow cytometer.
2. LIVE/DEAD Fixable Viability/Cytotoxicity kit for mammalian cells (ThermoFisher).
3. PE-conjugated anti-E-cadherin (Biolegend 324,106).
4. APC-conjugated anti-Fibronectin produced using the
Lightning-Link Rapid Conjugation System (Innova Biosciences) and anti-Fibronectin (Agilent A0245).
2.6 Cell Microscopy
1. Microscope equipped with phase-contrast and epifluorescence
transillumination.
2. Anti-β-Catenin (Cell Signaling Technology 8480S).
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Cecile Davaine et al.
alpha (hTNFα) (StemCell Technologies or any equivalent reference from other suppliers) at 50 μg/mL prepared following
providers’ recommendation) and stored at À20
C.
14. Control medium composed of DMEM/F12 supplemented
with 5% horse serum, 10 ng/mL cholera toxin, 10 μg/mL
insulin, 0.5 μg/mL hydrocortisone, 20 ng/mL human recombinant epidermal growth factor, 1% Penicillin/Streptomycin,
1% L-Glutamine (see Note 2).
15. EMT inducing medium (EMT-IM): control medium plus
TGF-β (5 ng/mL), TNF-α (10 ng/mL), and IL-6 (10 ng/
mL) (see Notes 2 and 3).
2.4 Real-Time PCR
1. Total RNA extraction kit (PureLink™ RNA Mini Kit, ThermoFisher) or any equivalent reference from other suppliers.
2. High-Capacity cDNA Reverse Transcription kit (ThermoFisher) or any equivalent reference from other suppliers.
3. FastStart Universal SYBR Green Master mix (Roche).
4. Real-time PCR primers:
SNAIL1: FW 5
0 -GCTGCAGGACTCTAATCCAGA-3
0 ; RV 50 -ATCTCCGGAGGTGGGATG-3
0 [11].
SNAIL2: FW 5
0 -TTCGGACCCACACATTACCT-3
0 ; RV 50 -TCTCCCCCGTGTGAGTTCTA-3
0 .
ZEB1: FW 5
0 - AGGGCACACCAGAAGCCAG-3
0 ; RV 50 -GAGGTAAAGCGTTTATAGCCTCTATCA-3
0 [12].
TBP: FW 5
0 -CACGAACCACGGCACTGATT-3
0 ; RV 50 -TTTTCTTGCTGCCAGTCTGGAC-3
0 [12].
36B4: FW 5
0 -GCTGATGGGCAAGAACACCA-3
0 ; RV 50 -CCGGATATGAGGCAGCAGTT-3
0 [11].
SMA: FW 5
0 -GAGCGTGAGATTGTCCGGGA-3
0 ; RV 5
0 -
AAGGGAGGATGAGGATGCGG-3
0 .
5. Real-time PCR thermocycler.
2.5 Flow Cytometry
1. Flow cytometer.
2. LIVE/DEAD Fixable Viability/Cytotoxicity kit for mammalian cells (ThermoFisher).
3. PE-conjugated anti-E-cadherin (Biolegend 324,106).
4. APC-conjugated anti-Fibronectin produced using the
Lightning-Link Rapid Conjugation System (Innova Biosciences) and anti-Fibronectin (Agilent A0245).
2.6 Cell Microscopy
1. Microscope equipped with phase-contrast and epifluorescence
transillumination.
2. Anti-β-Catenin (Cell Signaling Technology 8480S).
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