Journal: Cancers
Article Title: Heterogeneity in the Metastatic Microenvironment: JunB-Expressing Microglia Cells as Potential Drivers of Melanoma Brain Metastasis Progression
doi: 10.3390/cancers15204979
Figure Lengend Snippet: MBM-derived leukemia inhibitory factor (LIF) upregulates JunB expression in microglia via JAK/STAT3 signaling. ( A ) Western blot analysis of JunB and β-tubulin in microglia cells treated with MCM combined with STAT3 (S727) inhibitor (zoledronic acid), JAK inhibitor (baricitinib), JNK inhibitor (SP600125), MEK/ERK inhibitor (U0126), and DMSO as a control for 3 h. Representative blot and quantification of JunB expression are presented (normalized to cells treated with MCM + DMSO). Data are shown as mean expression + SEM of biological replicates. * p < 0.05, ** p < 0.01, *** p < 0.005. ( B ) Western blot analysis of JunB and β-tubulin expression in microglia cells treated with LIF (25 ng/mL), OSM (50 ng/mL), IL-4 (20 ng/mL), IL-6 (20 ng/mL), IL-15 (50 ng/mL), and IL-27 (20 ng/mL) for 3 h. Representative blot and quantification of JunB expression are presented. Data are shown as mean expression + SEM of biological replicates. * p < 0.05. ( C ) ELISA measurement of LIF in MCM. The bars represent the average cytokine concentration + SEM. ( D ) Western blot analysis of p-STAT3 (Tyr705) and STAT3 expression in microglia cells treated with LIF (25 ng/mL) for 10 min. Representative blot and quantification of p-STAT3 (Tyr705), STAT3, and β-tubulin expression are presented. Data are shown as mean expression + SEM of biological replicates. * p < 0.05. ( E ) Western blot analysis of JunB and β-tubulin expression in microglia cells treated with LIF (25 ng/mL) with or without baricitinib for 3 h. Representative blot and quantification of JunB expression are presented. Data are shown as mean expression + SEM of biological replicates. * p < 0.05. ( F ) Flow cytometry analysis of LIFR and gp130 expression in microglia cells. ( G , 1–4) Western blot analysis of JunB and β-tubulin in microglia cells treated with or without MCM with LIFR inhibitor (EC359) or DMSO as control for 3 h. Representative blot and quantification of JunB expression are presented. Data are shown as mean expression + SEM of biological replicates. * p < 0.05, ** p < 0.01. In all the experiments, unless indicated otherwise, microglia cells grown in starvation medium served as controls.
Article Snippet: For the estimation of intracellular OSM levels, MBM cells were plated and grown in starvation media for 72 h. The cells were then lysed as previously described [ ], and 2 μg of the lysates was used to determine the intracellular levels of OSM using the Human OSM DuoSet ELISA Development Kit (DY295, R&D Systems).
Techniques: Derivative Assay, Expressing, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Concentration Assay, Flow Cytometry
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