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. 2013 Feb 8;123(3):1348–1358. doi: 10.1172/JCI65416

Figure 4. Paclitaxel and TGF-β induce SMAD4-dependent expression of IL-8.

Figure 4

(A) SUM159 and BT549 cells were treated with 5 to 10 nM paclitaxel for 4 days. RT-qPCR analysis was performed to assess IL8 and GAPDH RNA levels (*P < 0.05, **P < 0.003). (B) SUM159 cells were treated with 2.5 ng/ml TGF-β with or without 5 μM LY2157299 and grown as mammospheres. Media was collected, and IL-8 protein levels were measured by ELISA; IL-8 levels were normalized to total protein (*P = 0.02). (C) SUM159 cells were transfected with control or SMAD4 siRNA and plated as mammospheres with or without TGF-β1. After 6 days, mammospheres and media were collected and analyzed for IL-8 levels by ELISA. IL-8 levels were normalized to total protein (*P < 0.001). (D) SUM159 cells were transfected with control or both CXCR1 and CXCR2 siRNA and plated as mammospheres with or without 2.5 ng/ml TGF-β1 for 6 days. Mammosphere number was then quantitated, as described in Methods (*P = 0.016). (E) SUM159 cells were transfected with control or SMAD4 siRNA. Forty-eight hours later, 10 nM paclitaxel was added for 24 hours before mRNA extraction and RT-qPCR using IL-8–specific primers (*P < 0.002, **P = 0.002). (F) RT-qPCR analysis of IL8 mRNA levels in SUM159 and BT549 cells treated with 5 μM LY2157299 and 5 nM paclitaxel (BT549 cells) or 10 nM paclitaxel (SUM159 cells) as indicated for 6 days (*P < 0.007, **P < 0.001). (G) Media from cells treated with paclitaxel with or without LY2157299 was collected and subjected to IL-8 ELISA assay. Raw IL-8 levels were normalized to cell number (*P < 0.001). Error bars indicate SEM.