Journal: ImmunoTargets and Therapy
Article Title: Kasugamycin Inhibits Melanoma Lung Metastasis and CHI3L1-Driven M2-Like Tumor-Associated Macrophage Differentiation
doi: 10.2147/ITT.S563951
Figure Lengend Snippet: KSM inhibits CHI3L1-stimulated M2 macrophage activation via EGFR. Bulk RNA-seq sequencing analysis on the differentiated macrophages from THP-1 cells was used to evaluate mRNA expression in the macrophage differentiation regulated by CHI3L1 and KSM. ( A ). Volcano plots showing differentially expressed genes regulated by CHI3L1. ( B ) Representative plots of the top 20 genes that are upregulated (> 2-fold) by CHI3L1 stimulation but downregulated (< 2-fold) by KSM treatment. ( C ) Representative mRNA expression of EGFR in differentiated macrophages with CHI3L1 and KSM treatment was detected by real-time qRT-PCR. ( D ) Representative immunoblots showing p-EGFR (Tyr1068) and total EGFR expression in differentiated macrophages with CHI3L1 and KSM. Right panel, densitometric quantitation on the blots of EGFR and p-EGFR. ( E ) Effect of EGFR inhibition on the expression of p-STAT3 and total-STAT3 in CHI3L1-stimulated, differentiated macrophages by gefitinib (Tocris Bioscience, #3000) treatment (1 μM, 72 hours). Right panel, densitometric quantitation on the blots of p-STAT3 and total STAT3. ( F ) Effects of EGFR inhibition on the expression of CD163 and CD206 in CHI3L1-stimulated, differentiated macrophages by gefitinib treatment (0.1 and 1 μM, 72 hours). Right panel, densitometric quantitation on the blots of CD163 and CD206. The values in ( C – F ) are the mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001 (One-Way ANOVA, multiple comparisons).
Article Snippet: Protein lysates (25 μg) from lung tissues or cells were subjected to SDS-PAGE, transferred to membranes, and immunoblotted with primary antibodies against CD163 (#PA5-109327, Thermo Fisher Scientific), CD206 (E6T5J, #24595S, Cell Signaling Technology), phosphorylated AKT (p-Akt) (193H12, #4058S, Cell Signaling Technology), and total Akt (11E7, #4685S, Cell Signaling Technology), phosphorylated EGFR (Tyr1068) (p-EGFR) (#44-788G, Thermo Fisher Scientific), EGFR (D38B1, #4267S, Cell Signaling Technology), phosphorylated Erk (p-Erk) (#9101S, Cell Signaling Technology), total Erk (#9102S, Cell Signaling Technology), phosphorylated STAT3 (Tyr705) (p-STAT3) (D3A7, #9145, Cell Signaling Technology), total STAT3 (79D7, #4904, Cell Signaling Technology) and b-actin (C4, #sc-47778 HRP, Santa Cruz Biotechnology).
Techniques: Activation Assay, RNA Sequencing, Sequencing, Expressing, Quantitative RT-PCR, Western Blot, Quantitation Assay, Inhibition
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