Journal: Journal of Innate Immunity

Article Title: GSK3β Inhibition Prevents Macrophage Reprogramming by High-Dose Methotrexate

doi: 10.1159/000526622

Figure Lengend Snippet: LPS activated MTX-treated M-MØ exhibit a functional proinflammatory profile. a Experimental design. Monocytes were untreated or exposed to 5 µM MTX at the beginning of the 7-day macrophage differentiation process with M-CSF and challenged with LPS (10 ng/mL) on day 7. Cells were assayed 3 h post-LPS stimulation on M-MØ and MTX-M-MØ. b Production of IL-10, IFNβ, and IL-6 by M-MØ and MTX-M-MØ challenged with LPS for 3 h as determined by ELISA. Mean ± SEM of 10–12 independent donors, each symbol represents a single donor (** p < 0.01, *** p < 0.001, paired t test). c Upper panel, scatter plot of RNAseq results showing gene expression changes 3 h post-LPS stimulation in MTX-M-MØ (LPS + MTX-M-MØ/LPS + M-MØ). The number of annotated genes whose expression is upregulated or downregulated 3 h post-LPS stimulation in M-MØ after 7 days of MTX treatment (adjusted p <0.05) is shown. Lower panel, relative level of expression of the indicated genes as determined by RNAseq on LPS + M-MØ and LPS + MTX-M-MØ, adjusted p value is indicated. d GSEA on the ranked comparison of the transcriptome of LPS + MTX-M-MØ versus LPS + M-MØ, using the genes significantly modulated by LPS in GM-MØ (GM-MØ-specific LPS-induced) and M-MØ (M-MØ-specific LPS-induced) as data set. The genes within the leading edge of each GSEA are indicated in online supplementary Table . e, f Immunoblot analysis of pERK, pJNK, and pp38 ( e ), pIRF3 and pSTAT3 ( f ) by monocytes differentiated with M-CSF in the absence or presence of MTX for 7 days and challenged with LPS for the indicated time points. Vinculin or GAPDH protein levels were determined as protein loading control.

Article Snippet: Protein detection was carried out using rabbit polyclonal antibodies against pp38, pJNK, and pERK (clones D3F9, 81E11 and D13.14.4E; Cell Signaling, 1/1,000), pIRF3 (clone 4D4G; Cell Signaling, 1/1,000), pSTING (clone D7C3S; Cell Signaling, 1/1,000), STING (clone D2P2F; Cell Signaling, 1/1,000), pCSF1R (clone 49C10; Cell Signaling, 1/1,000), CD209 (dsg-1, 1/1,000) [ ], MAF (sc-7866; Santa Cruz Biotech, 1/1,000), MAFB (clone O91E9; BioLegend, 1/1,000), and pSTAT3 (clone D3A7; Cell Signaling, 1/2,000), goat polyclonal against CSF1R (AF329; R&D Systems, 1/2,000), and mouse monoclonal antibody against human FOLR2 (FRβ, kindly provided by Dr. Takami Matsuyama [ ], 1/800).

Techniques: Functional Assay, Enzyme-linked Immunosorbent Assay, Gene Expression, Expressing, Comparison, Western Blot, Control