Breast cancer cells induce stromal fibroblasts to express MMP-9 via secretion of TNF- and TGF-ß

JCS02334 Supplementary Material

Supplemental Material and Methods

Control of inhibitor effects and Western Blotting

To show effectiveness of the inhibitors used above, dermal fibroblasts (DFs) were grown to subconfluence, serum starved overnight and pre-incubated for 30 minutes to 2 hours with the respective inhibitor or corresponding amounts of DMSO. Cells were then treated with TNF-a (20 ng/ml,), TGF-b1 (2.5 ng/ml,) or EGF (50 ng/ml,) for the times indicated in Fig. S1. Manumycin was added to fibroblasts grown to 80% confluence without further stimulation. Protein lysates were obtained, and electrophoresis using a Tris-glycine based precasted system (Novex, Invitrogen, Carlsbad, CA) and western blots performed as described earlier (Tian et al., 2003) using the antibodies listed in Tab.S1.

Files in this Data Supplement:

• Supplemental Table (Adobe PDF)
• Supplemental Figure 1 -

  Fig. S1. (A) In-situ zymography of a 4-day-day old coculture of dermal fibroblasts (DFs) and CA1a or the corresponding homotypic cultures using DQ collagen IV as substrate. Similarly to gelatin in-situ zymography (Fig. 2), the main collagenolytic activity localizes to the stromal partner. Scale bar, 80 mm. (B) In-situ zymography using DQ collagen IV of cocultures of CA1a cells and MMP2KO and MMP9KO or the corresponding wild type fibroblasts. Again, the main collagenolytic activity is localized to the stromal cells and there is a trend towards less collagen degradation in knockout fibroblasts. Scale bar, 80 mm. (C) Morphology of cocultures of DF and 10A, At.1k, and CA1h cells. Similarly to cocultures of DF and CA1a cells cultures organize into spindle shaped fibroblasts surrounding epithelial or tumor cell islets. Scale bar, 200 mm. (D) In-situ zymography using DQ gelatin of DF and cocultures of DF and human breast cancer cell lines of increasing malignancy; as a trend, gelatinolytic activity of the culture increases with increasing malignancy of the tumor cells, paralleling the results seen by gelatin zymography of culture supernantants. Scale bar, 80 mm.

• Supplemental Figure 2 -

  Fig. S2. Inhibitors were tested for their efficacy on DF. (A) Cells were incubated with the ALK5 inhibitor SB431542 (5 mM) for for 2 hours before stimulation with TGF-b (5 ng/ml) for 30 minutes. (B, C) Cells were incubated with (B) the ERK inhibitor PD98059 (25 mM) or (C) the JNK-inhibitor SP600125 (10 mM) for 2 hours before stimulation with TNF-a (10 ng/ml) for 15 minutes. (D) Cells were incubated with the p38 inhibitor SB203580 (20 mM) for 30 minutes before stimulation with TNF-a (10 ng/ml) for 15 minutes. (E) Cells were incubated with the PI3K inhibitor LY294002 (20 mM) for 30 minutes before stimulation with insulin for 30 minutes. (F) Cells were incubated with manumycin for 24 hours to inhibit Ras farnesylation. (G) Cells were incubated with AG1478 (10 mM) for 30 minutes before stimulation with EGF (50 ng/ml) for 10 minutes