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Fig. 4 | Breast Cancer Research

Fig. 4

From: Expression of functional toll like receptor 4 in estrogen receptor/progesterone receptor-negative breast cancer

Fig. 4

Toll-like receptor 4 (TLR4) silencing decreases endogenous levels of pro-inflammatory cytokines. a Effect of TLR2/4 silencing in breast cancer cells transfected with negative control (nc) siRNA, or siRNA directed against TLR2 mRNA (si#1 and si#2) or TLR4 mRNA (si#1 and si#2) was analyzed using quantitative real-time PCR; ***P <0.001 (analysis of variance (ANOVA)). b IL-6 (left) and IL-8 (right) ELISA on supernatants from MDA-MB-231 breast cancer cells transfected with nc siRNA, or siRNA directed against TLR2 mRNA (si#1 and si#2) or TLR4 mRNA (si#1 and si#2); n = 4. Error bars standard error of the mean (SEM); *P <0.05, **P <0.01, ***P <0.001 (ANOVA). c Boyden chamber migration assays. Migration of primary human myeloid cells towards supernatants from different cell lines indicated. Human primary peripheral blood mononuclear cells were isolated as previously described [48] and allowed to migrate through a Costar Transwell® Permeable Support 8.0-μm 24-well plate (Corning) to the supernatants of breast cancer supernatants cultured under serum-free conditions. Percentage of migrated CD11b+ cells was analyzed using a flow cytometer and CD11b-APC antibodies (BD Sciences); n = 4. Error bars SEM; *P <0.05, **P <0.01, ***P <0.001 (ANOVA). d Matrigel invasion assays. Invasion of lipopolysaccharide (LPS)-stimulated/un-stimulated MDA-MB-231 cells into matrigel invasion chambers (BD Sciences) as indicated: 25 × 103 MDA-MB-231 cells were stimulated or not with LPS and allowed to invade from 72 h. Amount of invaded cells was analyzed using crystal violet staining and manual counting in four separate experiments; n = 4. Error bars SEM; *P <0.05, **P <0.01, ***P <0.001 (Student’s t test)

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