Fig. 4

CCL2 is required for LNMAT1-induced LN metastasis. a, b Heatmap representing unsupervised hierarchical clustering of genes regulated by LNMAT1 based on next-generation sequencing (NGS) analysis. Rows represent probe sets, and columns represent samples treated as indicated. Green, downregulation; red, upregulation. CCL2 is indicated by black arrows. c, d Bio-Plex multiplex immunoassay analysis of protein expression of 40 cytokines and chemokines regulated by LNMAT1. Two-tailed t-tests was used. e, f qRT-PCR of CCL2 expression in LNMAT1-transduced, LNMAT1-silenced and control cell as indicated. Statistical significance was assessed using one-way ANOVA followed by Dunnett’s tests for multiple comparison and two-tailed t-tests. g, h ELISA of CCL2 expression in the indicated cells. Statistical significance was assessed using one-way ANOVA followed by Dunnett’s tests for multiple comparison and two-tailed t-tests. i Representative images of popliteal LNs, enucleated LNs and IHC staining with anti-luciferase antibody in the indicated mice group (n = 16 per group). Scale bars: black, 500 µm; red, 50 µm. j, k The ratios of the metastatic to volume quantification (j) and Kaplan–Meier survival analysis (k) for the indicated group (n = 16 per group). Two-tailed t-tests was used. l Representative images of HE and IHC staining confirming the LN status (n = 16). Scale bars: black, 500 µm; red, 100 µm. m Volume quantification of popliteal LN metastasis after shRNA-mediated depletion of CCL2. One-way ANOVA followed by Dunnett’s tests for multiple comparison was used. The error bars represent standard deviations of three independent experiments. *p < 0.05 and **p < 0.01