1. - + - + - + Butler et al Supplementary Figure 1 A B C D - E BL21
BL21+VTSP
VTSP
[M+H]+
10,837 Da A 40 25 20 50 15
kDa B
kDa 45 29 18 14
[His6]-GQGGGL - +
DTT - +
DTT
kDa 45 29 18 14 C
[M+H]+
19,286.9 Da D
kDa 29 18 14 - +
DTT
MMP - 1 8 13 2 9 3 14 15 7 E
kDa 4 25 16
Silver stain 20 40 30
α-VTSP
Supplementary Fig 1: Expression and processing of recombinant CCN5 variable region and thrombospondin domains (VTSP). A) cDNA encoding human CCN5 residues Gly164-Phe250 was subcloned into pGYMX and expressed in BL21 Gold (DE3) E. coli. Recombinant VTSP was purified and refolded from inclusion bodies. The m/z [M+H]+ of the VTSP was measured by MALDI-TOF MS on a Voyager-DE STR (Applied Biosystems) as 10,837.0 Da. This deconvolutes to an N-terminus without the initiating methionine (predicted m/z [M+H]+ 10,839.8). Following refolding, the mobility of B) VTSP, C) N-CCN5, or D) C-CCN5 ± dithiothreitol (DTT) was assessed to confirm disulphide bond formation. The N-terminal sequence of VTSP determined by Edman degradation is shown. The m/z [M+H]+ of N-CCN5 was measured as 19,286.9 as above. This deconvolutes to an N-terminus without the initiating methionine (predicted mass 19,289.12). All products were visualised by Coomassie Brilliant Blue staining after 15% SDS-PAGE.
E) Recombinant VTSP was incubated for 18 h at 37 °C (molar ratio 10:1 VTSP:MMP) and analysed on 15% Tris-Tricine SDS-PAGE gels by silver staining and western blotting using affinity purified anti-VTSP rabbit polyclonal antibody. All MMPs were human except for rat MMP7.

2. Butler et al Supplementary Figure 2
MMP14
CCN5
MMP14/CCN5
control
hours 24 48
Transfected cDNA
Supplementary Fig 2: RT-PCR analysis of CCN5 and MMP14 expression in MCF7 cells transfected with MMP14, CCN5, or both cDNAs, or control vectors after 24 and 48 h. Total RNA was extracted from cells using the High Pure RNA Isolation Kit (Roche Molecular Biochemicals, IN, USA). RT-PCR amplification was performed using the GeneAmp Thermostable rTth reverse transcriptase RNA PCR kit (Applied Biosystems, NJ, USA) following manufacturer’s instructions. Reverse transcription was carried out with 5’-AGGTCTCCCCTTCCCGATACA-3’ as the CCN5 primer, 5’-CCATTGGGCATCCAGAAGAGAGC-3’ as the MMP14 primer or
5’-GATTCTGACTTAGAGGCGTTCAGT-3’ as the 28S primer for 15 min at 70 °C. PCR products were generated with these same primers and with forward primers
5’-CCTCCTCTGCCTCCTCTCAAA-3’ for CCN5, 5’GGATACCCAATGCCCATTGGCCA-3’ for MMP14 or 5’-GTTCACCCACTAATAGGGAACGTGA-3’ for 28S as loading control. PCR conditions were 95 °C for 2 min, followed by 35 cycles for CCN5 consisting of 94 °C for 20 sec, 55 °C for 20 sec, 72 °C for 30 sec and a final elongation step of 72 °C for 2 min. For MMP14 30 cycles and 28s 18 cycles consisting of 94 °C for 20 sec, 66 °C for 20 sec, 72 °C for 30 sec and a final elongation step of 72 °C for 2 min. Amplification products were electrophoresed on agarose gels, stained with Gelstar (Sanver Tech, Belgium), scanned with a FluorSImager, and analysed using Multianalyst software (Biorad, Belgium). RT-PCR confirms expression of significant amounts of mRNA for CCN5 and MMP14, only when the MCF-7 cells were transfected with the relevant cDNA.