Journal: Nature

Article Title: Tumoural activation of TLR3-SLIT2 axis in endothelium drives metastasis

doi: 10.1038/s41586-020-2774-y

Figure Lengend Snippet: a, Primary MLECs (ICAM2-positive) upregulate SLIT2 when treated with conditioned medium derived from 4T1 cells (n = 3). Dot plot represents Slit2 mRNA levels measured by qPCR for each biological replicate with mean ± s.e.m. Two-tailed Student’s t-test. b, Primary nonendothelial cells (ICAM2-negative) from the lung do not upregulate SLIT2 upon treatment with 4T1 conditioned medium (n = 3). Dot plot represents Slit2 mRNA levels measured by qPCR for each biological replicate with mean ± s.e.m. Two-tailed Student’s t-test. c, Treatment of endothelial cells with 5 μM dynasore inhibits SLIT2 expression upon treatment with conditioned medium from 4T1 cells (n = 3). Dot plot represents Slit2 mRNA levels measured by qPCR for each biological replicate with mean ± s.e.m. Two-tailed Student’s t-test. d, e, Dot plots represent Slit2 mRNA expression by qPCR in endothelial cells exposed to 4T1 conditioned medium treated with (e) DNase I (10 μg/ml; n = 3), and (d) heat treatment (95 °C, 10 min; n = 3). Data are mean ± s.e.m. Two-tailed Student’s t-test. f, TLR3 wild-type (Tlr3 WT) and TLR3-knockout (Tlr3 KO) endothelial cells were treated with conditioned medium from 67NR, 4T07 and 4T1 cells. Western blot analysis revealed that wild-type endothelial cells display increased phosphorylation of ERK1 and ERK2 upon treatment with the conditioned medium from highly metastatic 4T1 cells. TLR3-knockout endothelial cells displayed reduced phosphorylation of ERK1 and ERK2 relative to wild-type controls. Dot plot displays densitometry quantification for three independent experiments. Two-tailed Student’s t-test. g, RNase A treatment of the 4T1 conditioned medium blunted endothelial phosphorylation of ERK1 and ERK2. h, Supplementation of basal medium with synthetic TLR9 ligand (CpG ODN, 2.5 or 12.5 μg/ml) did not induce endothelial SLIT2 upregulation (n = 3). Dot plot represents Slit2 levels measured by qPCR for each biological replicate with mean ± s.e.m. Two-tailed Student’s t-test. i, j, Supplementation of basal medium with synthetic TLR9 ligand (CpG ODN, 2.5 or 12.5 μg/ml) induced (i) endothelial Il6 (n = 3) and (j) Ifng mRNA expression (n = 3). Dot plot represents Il6 and Ifng levels measured by qPCR for each biological replicate with mean ± s.e.m. Two-tailed Student’s t-test. k, l, Quantification of RNA isolated from conditioned medium of (k) B16F0 (n = 3) and B16F10 cells (n = 3) and (l) 67NR (n = 3) and 4T1 cells (n = 3). Dot plot represents RNA concentrations detected in conditioned medium normalized by the cell number with mean ± s.e.m. Two-tailed Student’s t-test. m, RNA detection in plasma isolated from mice with 67NR (n = 3) and 4T1 (n = 5) mammary gland tumours. Tumour-free mice (n = 5) were used as a negative control. Increased concentrations of RNA were detected in the plasma of mice with the metastatic 4T1 tumours. Dot plot represents the RNA concentrations detected in the plasma of each mouse, either with no tumour or with 67NR and 4T1 tumours. Two-tailed Student’s t-test.

Article Snippet: Two-tailed Student’s t -test. g , RNase A treatment of the 4T1 conditioned medium blunted endothelial phosphorylation of ERK1 and ERK2. h , Supplementation of basal medium with synthetic TLR9 ligand (CpG ODN, 2.5 or 12.5 μg/ml) did not induce endothelial SLIT2 upregulation ( n = 3).

Techniques: Derivative Assay, Two Tailed Test, Expressing, Knock-Out, Western Blot, Phospho-proteomics, Isolation, RNA Detection, Clinical Proteomics, Negative Control