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. 2022 Feb 15;13:879. doi: 10.1038/s41467-022-28531-1

Fig. 5. TLR4 regulates the altered phenotype of mdx BMDM.

Fig. 5

a Basal M1 and M2 marker gene transcript levels (expressed relative to the mean age-matched WT level) in BMDM from WT, mdx, mdxTLR4–/– mice at different phases of disease (n = 4 for iNOS in prenecrotic phase and IL12α in necrotic phase mdx groups, rest n = 5/group). b Heatmap showing unsupervised hierarchical clustering analysis of samples based on the gene expression data shown in a. Genes with higher and lower expression levels are identified as red or blue (variable units proportional to color intensity), respectively, whereas outlier samples are black. c, d Oxygen consumption rate (OCR) data for mdx and mdxTLR4-/- BMDM at necrotic (n = 5/group) and fibrotic phases (n = 5/group) of the disease. e–h WT, mdx, and mdxTLR4–/– BMDM at necrotic phase were stimulated with LPS + IFNγ (n = 5/group) or IL4 (n = 5/group) or fibrinogen (n = 3/group) to determine mRNA transcript levels (expressed relative to the mean WT-unstimulated value) of: (e, g) M1 and (f, h) M2 marker genes. i western blot images and densitometric analyses of total iNOS and Arginase1 in cell lysates of fibrinogen-stimulated (24 h) WT, mdx and mdxTLR4–/– BMDM from necrotic phase mice (n = 3/group; all experimental replicates are shown). j ELISA measurements of TNF and IL6 protein levels in culture supernatants from WT, mdx and mdxTLR4–/– BMDM after stimulation with fibrinogen for 24 h (n = 4/group). k, l As shown in Fig. 4a, WT and TLR4-/- BMDM were trained with mdx-ME and secondarily challenged with k fibrinogen (8 h; n = 4/group) or l β-glucan (4 h; n = 4/group), followed by qPCR. The graph shows mRNA transcript levels of genes relative to the mean control (PBS-trained and RPMI-stimulated) WT group. Data represent means ± SEM of biologically independent samples from different mice; a *P < 0.05 WT vs. mdx and †P < 0.05 mdx vs. mdxTLR4–/– (one-way ANOVA followed by Tukey post-hoc test, two-tailed); c, d *P < 0.05 WT vs. mdx (unpaired t-test, two-tailed); e–j *P < 0.05 vs. WT and †P < 0.05 vs. mdx at a given time point (one-way ANOVA followed by a Tukey post-hoc test, two-tailed); k *P < 0.05 PBS vs. ME and †P < 0.05 WT vs. TLR4–/– group (two-way ANOVA followed by a Tukey post-hoc test, two-tailed). See Source Data file for the exact P-values.