Figure 2.

IL1β induces TSLP production from breast cancer cells. A, IL1β, TSLP, IFNγ, and IL13 in supernatants of breast cancer samples. The logarithm of four cytokine measurements (IL1β, TSLP, IL13, IFNγ: left to right at x-axis and top to bottom at y-axis) was taken for total 145 patients and the dependency between any pair of cytokine measurements was tested under the null hypothesis of independence among the four measurements. Log-likelihood and its Bartlett correction were performed and P values for both methods were significantly low (P < 0.001). Histogram plots indicated distribution of the cytokines expression among all patients. Pairwise correlations were tested among 6 pairs of cytokine measurements. Scatter plots indicated the correlation pairwise. Using Pearson and Spearman correlation, all P values were found significant (P < 0.025). B, Heatmap generated on the basis of the log-transformed four cytokine measurements using R package “pheatmap” with “ward.D2” method. Patients were split into four groups based on the “ward.D2” clustering method. The levels of cytokines were color coded as indicated. C, RNA transcripts visualized in breast cancer tumor sections with QuantiGene ViewRNA ISH. Human KRT8 and human TSLP are green and red, respectively. D, MDA-MB-231 cells were treated with medium alone, 10 ng/mL of IL1β, IL1α, TNFα, or IL6 for the indicated time course. TSLP mRNA level by quantitative real-time PCR normalized to internal control GAPDH. Bars show the mean ± SEM for triplicate wells from a representative experiment. *, P < 0.05; **, P < 0.0i; ***, P < 0.000i. E, ChIP by using anti-RNA polymerase II was performed. ChIP-qPCR analysis on lfTSLP (black) and sfTSLP (white) genes from MDA-MB-231 cells with IL1β or IL1β blocking for 1 hour. Percentage of input summarized from three experiments.`