Figure 4. Tumor EVP-packaged palmitic acid induces TNFα secretion from KCs and promotes fatty liver generation.

a, TNFα concentration determined by ELISA in the plasma from B16F10-Tb (left) and K7M2-Tb mice (right), and PBS-injected controls. B16F10-Tb model (n=6 controls, n=9 Tb mice), K7M2-Tb model (n=5). b, TNFα concentration in the plasma of B16F10-CL-EVP- and B16F10-TE-EVP-educated mice (n=4 each) (left), K7M2-CL-EVP- and K7M2-TE-EVP-educated mice (n=5 each) (right), and PBS-educated controls. c, Representative images (left) and associated statistical analysis (right) of BODIPY staining of the primary hepatocytes 48 h post treatment with recombinant murine TNFα protein (25 ng/ml), or PBS control. n=7 independent experiments. d,e, Statistical analysis of BODIPY staining of the livers from B16F10- and K7M2-Tb mice (d), and B16F10-CL-EVP- and K7M2-CL-EVP-educated mice (e), treated with anti-TNFα antibody or IgG1 isotype control. B16F10-Tb model (n=7), K7M2-Tb model (n=3), B16F10-CL-EVP model (n=5), K7M2-CL-EVP model (n=4). f, g, Quantitative analysis of the long-chain free fatty acids in the EVPs derived from B16F10 and Melan-a cells (f), and K7M2 cells and primary murine osteoblasts (g). n=5 per group. h, qRT-PCR analysis of the Tnf expression in KCs 4 h post treatment with PBS, 10 μg of EVPs derived from DMSO or C75 (40 μM) treated B16F10 cells (left) or K7M2 cells (right). n=4 independent experiments. i, qRT-PCR analysis of Tnf expression in KCs which were pre-treated with DMSO or TAK (5 μM) for 1 h, and subsequently treated with PBS, 10 μg of B16F10-CL-EVPs (left) or K7M2-CL-EVPs (right) with or without TAK (5 μM) for 4 h. n=3 independent experiments. P values were determined by the two-tailed, unpaired Student’s t-test (a-g), or one-way ANOVA with Tukey’s test (h,i). Data are mean ± s.e.m. Scale bar, 20 μm. NS, not significant.