Fig. 3.

S100A9 promotes the accumulation of fatty acids in EGFR-wild-type tumor cells. A Nimotuzumab and Tocilizumab reduced the elevated intracellular FFA levels induced by mtLC-MSCs. B Transcriptional S100A9 regulated by conditioned medium. C Indicated protein levels under conditioned medium treatment. D mtLC-MSC-induced upregulation of S100A9 transcription was reduced by nimotuzumab and tocilizumab. E S100A9 accumulated in nucleus after mtLC-MSC-conditioned medium treatment. F, G S100A9 secretion with conditioned medium (F) and blockaded by nimotuzumab and tocilizumab (G). H, I Downstream pathways activated by conditioned medium treatment (H) and inhibited by nimotuzumab and tocilizumab (I). J STAT3 inhibitor stattic decreased mtLC-MSC-induced S100A9 transcription. K ChIP-qPCR confirmed STAT3 binding sites in the S100A9 promoter. L S100A9 silencing attenuated intracellular FFA levels. M, N S100A9 silencing reduced transcriptional (M) and protein (N) levels of downstream targets. O Blockade of S100A9 receptors TLR4 and RAGE attenuated protein levels of downstream targets. Conditioned medium treatment was applied for 72 h (A–F), 30 min (H, I), or 24 h followed by normal medium for silencing (L-N). Blocking antibodies and inhibitors were pre-treated for 1 h. Nimotuzumab (20 µg/mL), Tocilizumab (100 ng/mL), Stattic (10 µM), rhS100A9 (500 ng/mL), FPS-ZM1 (10 µM), and TLR4-IN-C34 (10 µM). Statistical results were shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001. One-way ANOVA test except panel J and K (Student’s t-test). FFA, free fatty acid; GLUT1, glucose transporters 1; FASN, fatty acid synthase; TLR4, toll-like receptor 4; RAGE, receptor for advanced glycation end products