Figure 2. HS5-CM suppression of MCF7 growth is dependent on IL1.

A. IL1RA reversal of HS5-CM mediated growth suppression as shown by crystal violet staining. MCF7 cells were stained on 15 day post indicated treatment. B. Blockade of IL1 signaling by IL1RA partially reverses cell cycle effects of HS5-CM. Cells were treated with 1uM nocodazole for 24 hrs prior to analysis. Data are expressed as mean +/− SD of 3 independent experiments (day 5 n=2, day 7 n=1). There was no significant difference between any of the groups in the sub G1 phase. In S phase only the comparison between control and HS5-CM treated cells was significant, p=0.040. All groups except for control compared to IL1 RA were significantly different in the G0/G1 and G2/M phases (one way Anova, Tukey post-test correction). C. Immunofluorescence detection of IL1β (green) and IGF-1 (red) in stromal cells. D. Effect of stromal CM’s on MCF7 growth, with or without IL1-signaling blockade. Cells were treated on days 0 and 3 with IL1 β (100 pg/ml) or with 30% CM from indicated stromal cells (adjusted to 1 ml CM per 500K stromal cells) with or without IL1RA (50 ng/ml) as indicated. 3–4 independent experiments shown, see Supplemental methods for analysis. E. Growth at 7 days of MCF7 or MDA-MB-231 cells exposed to HS5- or hFOB CM (20%) or medium (DMEM, DMEM/F12 respectively) alone, or to 100pg/ml or 100 ng/ml IL1β. Comparisons for each cell line by 1-way ANOVA with Tukey’s correction for multiple comparisons. Two independent experiments. *p<0 .05, **p<0.01, ***p<0.001.