Supplementary Materials and Methods
S100A2 expression in breast tumours: Publically available datasets (1-3) with
BRCA1 mutation/methylation status were analysed using Oncomine for S100A2
expression.
Correlation of S100A2 expression and 17-DMAG IC50 values: 17-DMAG IC50
values for a range of cell lines was extrapolated from Lehmann et al (4). Using the
publically available datasets used within this paper (GSE10890 and E-TABM-157),
microarray data from cell lines was analysed for S100A2 expression using R.
Samples were background-corrected, normalised and transformed using the Affy
package, justRMA. The probeset for S100A2 was identified and the relevant
expression levels determined. Correlation between S100A2 and 17-DMAG IC50
values was assessed by Spearman Correlation in Prism.
Real time Primers:
S100A2: F 5’-gctctccttcctgggtctgt-3’ and R 5’ – tgctccagagaactgcacat -3’, p53 F 5’tccgagtggaaggaaatttgc-3’ and R 5’-gatggtggtacagtcagagcc-3’, Cloning full length
Flag-tagged S100A2 F 5’ –gccatggactacaaggacgacgatgacaagatgtgcagttctctggag-3’ and
R 5’ –gcttcagggtcggtctg-3’
All other primers have been previously described (5-8).
Antibodies: S100A2: AF4870 R and D Systems, p53: FL-393 (sc6243) and D0-1
(sc126) Santa Cruz Biotechnologies Inc, HSP90 (sc7947) and HOP(sc27962) Santa
Cruz Biotechnologies Inc. All other antibodies has been previously described (6).
siRNA
S100A#2 ACAAGTTCAAGCTGAGTAA
S100A2#3 GCCAAGAGGGCGACAAGTT
All other siRNA has been previously described (6)
P63 overexpression constructs have been previously described (6).
P63 C40 luciferase construct has been descibed previoulsy (6)
Deletion and Mutation of S100A2 Luciferase construct: Using the p53/p63 binding
sites described in (9), the first p53 site was deleted using the restriction enzymes
Xho1 and Nde1 followed by religation. SDM to mutate the second site was carried
out using sequential SDM with primers
p53SDM1 F 5’ CAGGGTTTGGTGGGATTGGATTGAGGCAGGTTTGGT-3’ and
R 5’-ACCAAACCTGCCTCAATCCAATCCCACCAAACCCTG-3’ and p53SDM2
F 5’-TGGGATTGGATTGAGGTGGATTTGGTTTCCTTAAAA-3’ and R 5’TTTTAAGGAAACCAAATCCACCTCAATCCAATCCCA-3’. The mutated bases
are in bold.
References for Supplementary Materials and Methods
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