Fluorescent Microsatellite Genotyping of Cell Lines

Cell pellets were resuspended in PBS to a final volume of 200µl. DNA was prepared using a QIAamp DNA mini kit (QIAGEN Ltd, UK) using 100µl sterile distilled water in the final elution stage. DNA was quantified using a nanodrop spectrophotometer and diluted to a final concentration of 20ng/µl. 1µl of DNA from each cell line was then amplified using a panel of fluorescently labelled primer pairs for ten independent highly polymorphic microsatellite markers. PCR was performed in a volume of 25µl, containing a final concentration of 1.5mM MgCl2, 0.2mM for each dNTP, 0.4mM for each primer, and 1 unit of Taq polymerase (Fermentas, UK). Amplification was carried out with an initial denaturing step of 95oC for 3 minutes, followed by amplification for 30 cycles at 95oC for 1 minute, 55oC for 1 minute and 72oC for 2 minutes and a final extension step at 72oC for 8 minutes. PCR products were diluted as appropriate and subsequently resolved by capillary electrophoresis, using an ABI PRISM 310 Genetic Analyser (Applied Biosystems, Ltd). Analysis and sizing of microsatellite polymorphisms was performed using ABI PRISM GeneScan software (Applied Biosystems, Ltd).

RT-PCR Primers

ER Forward5’-CAGATGGTCAGTGCCTTGTTGG-3’

ER Reverse5’-CCAAGAGCAAGTTAGGAGCAAACAG-3’

PR Forward5’-CCAGAGCCCACAATACAGCTTC-3’

PR Reverse5’-GCCAGCCTGACAGCACTTTCTAAG-3’

pS2 Forward5’-TTGGAGCAGAGAGGAGGCAATG-3’

pS2 Reverse5’-TAGGATAGAAGCACCAGGGGACC-3’

GAPDH Forward5’-TCC CAT CAC CAT CTT CCA-3’

GAPDH Reverse5’-CAT CAC GCC ACA GTT TCC-3’

Syber-Green labelled Real-time RT-PCR Primers (Primer Design Ltd, UK)

BCL2 Forward5’-GAGGTCACGGGGGCTAATT-3’

BCL2 Reverse5’-GAGGCTGGGCACATTTACTG-3’

BID Forward5’-CTGTAAACTTCCAATTTCTAGCCATA-3’

BID Reverse5’-GTCCAACTGCTCTTTCATCTTTT-3’

BIK Forward5’-TGAAGTAAGACCCCTCTCCAG-3’

BIK Forward5’-CCAAAGAATCGAAGTCCTCCAT-3’

BAD Forward5’-CCTGGTGGGATCGGAACTT-3’

BAD Reverse5’-TGGGAACGGGGTGGAGTTTC-3’

GAPDH Assay number HK-SY-hu-600 (Primer design Ltd.)

Taqman Gene Expression Assays (Applied Biosystems)

Gene Name:ABI Taqman Assay Number

ER (ESR1)Hs00174860_m1

GAPDHHs99999905_m1

TFF1 (pS2)Hs00170216_m1

PRHs00172183_m1

CTDHs00157201_m1

GREB1Hs00536409_m1

NCOA1 (SRC1)Hs00186661_m1

NCOA2 (TIF2)Hs00896106_m1

NCOA3 (AIB1)Hs00180722_m1

NCOR1 (NCoR)Hs00196920_m1

NCOR2 (SMRT)Hs00196955_m1

EGFRHs00193306_m1

ERBB2 (HER2)Hs00170433_m1

CCND1 (cyclin D1)Hs00277039_m1

MYCHs00153408_m1

Antibodies

ProteinDilutionSupplierCatalogue Number

ER1:2000NovacastraNCL-L-ER6F11

P-S118 ER1:1000NEB2511

P-S167 ER1:1000NEB2514

PR1:250Santa CruzSC-538

pS2 (TFF1)1:500Santa CruzSC-8925

CTD1:3000Abcamab6313

MYC1:1000Santa CruzSC-788

CCND1 (Cyclin D1)1:1000Abcamab24249

ß-actin1:50,000Abcamab6276

GAPDH1:1000Abcamab9484

BCL21:1000Abcamab692

BIK (NBK)1:200Santa Cruzsc-10770

BID1:200Santa Cruzsc-11423

BAD1:200Santa Cruzsc-8044

EGFR1:250Santa CruzSC-03

MAPK1:1000NEB9102

P-MAPK1:1000NEB9106

AKT1:1000NEB9272

P-AKT1:1000NEB9271

P-HER2 (Y1248)/1:1000NEB2244

EGFR (Y1173)

NCOA1 (SRC1)1:1000Abcamab84

NCOA2 (TIF2)1:1000Abcamab10491

NCOA3 (AIB1)1:250BD Biosciences611105

NCOR1 (NCoR)1:1000Abcamab24552

NCOR2 (SMRT)1:250BD Biosciences611387

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