Carcinoma and stromal enzyme activity profiles associated with breast tumor growth in vivo

Jessani et al. 10.1073/pnas.0404727101.

Supporting Information

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Supporting Text
Supporting Figure 7
Supporting Table 1
Supporting Figure 8
Supporting Figure 9
Supporting Figure 10
Supporting Figure 11

Supporting Figure 7

Fig. 7. Serine hydrolase (SH) activity profiles of the membrane proteomes of MDA-MB-231 xenograft tumors and mouse mammary fat pad (mfp). Samples were processed and analyzed as described in the text for soluble proteomes. Note that the integral membrane SH activity KIAA1363, previously shown to be up-regulated in invasive cancer lines (1), is highly expressed in MDA-MB-231 xenografts.

1. Jessani, N., Liu, Y., Humphrey, M. & Cravatt, B. F. (2002) Proc Natl. Acad. Sci. USA 99, 10335–10340.

Supporting Table 1

Table 1. Tryptic peptide maps of probe-labeled enzyme activities. This table provides examples of representative tryptic peptides obtained from MS analysis that facilitated the distinction of tumor (human)-derived serine hydrolase activities from those arising from the stroma (mouse). Asterisks indicate peptides shared between homologous enzymes (e.g., CE-1 and MGC18894). Also see text for details.

Supporting Figure 8

Fig. 8. Down-regulation of specific sulfonate ester (SE)-reactive enzyme activities in mfp-cultivated MDA-MB-231 cells (231mfp cells). (A) Representative in-gel fluorescence analysis of SE-reactivity profiles of the soluble proteomes of parental MDA-MB-231 and 231mfp cells showing the down-regulation of platelet-type phosphofructokinase (PFK) and select other SE-labeled proteins (double arrowheads). (B) Quantification of levels of PFK and GSTw as measured by in-gel fluorescence scanning, showing the selective down-regulation of the former enzyme in 231mfp cells. Data reported as averages ± standard errors (SE); n = 3 per group. (C) Relative mRNA levels for PFK as measured by Northern blotting (n = 3 per group; values expressed in arbitrary units normalized to an internal 7S RNA control).

Supporting Figure 9

Fig. 9. Comparison of the SH activity profiles of the soluble (cytosol) and particulate (membrane) fractions of parental MDA-MB-231 and 231mfp cells. Note the high similarity between the parental and 231mfp profiles, which contrasts sharply with the large differences observed in the corresponding secreted proteomic profiles of these cells (Fig. 4).

Supporting Figure 10

Fig. 10. Relative mRNA levels for plasminogen activator inhibitor-1 (PAI-1) as measured by Northern blotting (n = 3 per group; values expressed in arbitrary units normalized to an internal 7S RNA control).

Supporting Figure 11

Fig. 11. Coomassie blue-stained protein gel of soluble mfp and xenograft tumor samples analyzed by activity-based protein profiling (corresponding enzyme activity profiles shown in Fig. 1).