Extended Data Fig. 6: Functional characterization of mIL-4 and mNeo-4 in mouse volumetric muscle loss model.

a, Illustration of experimental design and dosing regimen. Mice were treated with proteins or PBS for 4 consecutive days. Treatments were injected directly at the injury site on day 0, and the subsequent 3 treatments were injected subcutaneously. b, qRT-PCR analysis of genes related to tissue regeneration in isolated muscle samples (n = 1) from Pilot Study 1 (1.5 μg of mIL-4 or PBS treatments). Effects of mIL-4 were normalized to the PBS control. Fold change (FC) represents 2^-ΔΔCT. c, Repeated qRT-PCR analysis of M2-like macrophage-related genes in isolated muscle samples (n = 4) from Pilot study 1. d, qRT-PCR analysis of M2-like macrophage-related genes from the isolated muscle samples (n = 5) in Pilot Study 2 (1.5 μg of mIL-4, 1.5 μg mNeo-4, or PBS treatments). Effects of mIL-4 or mNeo-4 were normalized to the PBS control. e-f, Mice were treated with either mNeo-4 (at low [0.03 µg, n = 5], medium [1.6 µg, n = 5], or high [71 µg, n = 4] dose), 1.5 µg mIL-4 (n = 5), or PBS (n = 5) in the model shown in a. qRT-PCR analysis of M2-like macrophage-related genes in isolated (e) muscle and (f) spleen samples are shown. Effects of each treatment were normalized to the PBS control. Data represent mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, two-way ANOVA. For ease of visualization, only significance compared to the control cohort is shown in panels c-f. All p-values are recorded in Supplementary Table 7. g, NanoString analysis (myeloid panel) on the muscle samples from mice treated as described in e-f. Volcano plots of differentially expressed genes are shown for the mNeo-4 versus the mIL-4 cohort. The significance of differential gene regulation was determined by calculating adjusted p values using the Benjamini-Yekutieli method of estimating false discovery rates (FDR) in the nCounter software (thresholds of adjusted P < 0.05, and 0.01 are shown).

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