Fig 2: Microglia-derived IGF-1 is a potent mitogen for astrocytes and inducer of astrocytic migration towards an injured area. a–d In situ hybridization (ISH) signal for the proinflammatory cytokines IL-1a, IL-1ß, IL-6, and TNF in the injured mouse spinal cord (lesion epicenter) at 7 days post-SCI (dpi). e–g, i–k Representative darkfield photomicrographs showing expression of Tgfb1 and Igf1 mRNAs at the lesion epicenter at 7 dpi in C57BL/6 mice fed the control diet, PLX73086 or PLX5622. h, l Quantification of ISH signal (in mean grey values, MGV) for TGF-ß1 (h) and IGF-1 (l) at the lesion epicenter in mice treated with vehicle (Control, blue bars), PLX73086 (red bars) or PLX5622 (green bars) (n = 6 per group). m Quantification of the number of BrdU+ YO-PRO-1+ nuclear profiles following treatment of primary astrocyte cultures with either TGF-ß1, IGF-1 or control solution (n = 6 per group). n Quantification of the wound closure response in the different groups (n = 6 per group). o–q Representative immunofluorescence images showing the expression of IGF-1 (green signal, o, p) by TdT+ microglia (red cells, o and q) accumulating at the lesion border at 7 dpi. White arrowheads indicate co-localization of IGF-1, TdT, and DAPI (blue). r Quantification of Sox9+ astrocytes, expressed as the AUC of the total number of Sox9+ cells (×103 per mm3) from 800 µm rostral to 800 µm caudal to the epicenter, in the injured spinal cord of C57BL/6 mice treated with the IGF-1R antagonist OSI-906 (red bar) or the vehicle solution (Control, blue bar) (n = 4 per group). Data are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, compared to the control group. Statistical analysis was performed using either a one-way (h, l, and m) or two-way (n) ANOVA followed by a Bonferroni’s post hoc test, or a Student’s t-test (r). Scale bars: (a–g and i–k, in k) 200 µm, (o–q, in q) 20 µm

Fig 3: Pro-resolving factors released by macrophages after efferocytosis include TGF-ß, IGF-I and VEGF and their capacity to attract primary fibroblasts and IEC are dependent on these growth factors. (A) EGF, FGF, TGF-ß, IGF-I and VEGF were quantified by ELISA in the supernatants of macrophages after efferocytosis (SuperMApo) and in the control supernatants of macrophages (MacroSup) cultured alone or apoptotic cells (ApoSup) cultured alone. Data represented as mean ± s.e.m. are the pool of six independents experiments; *p < 0.05; ***p < 0.001 (one-way ANOVA with Tukey’s multiple comparison post-test). (B) Proliferation of mouse primary fibroblasts was assessed by MTT assay for 4 days with or without neutralizing antibodies to TGF-ß, IGF-I or VEGF, or control isotype. Data are from one representative experiment out of three and expressed as mean + s.e.m. of 3 replicates per condition; *p < 0.05; **p < 0.01, ****p < 0.0001 (two-way ANOVA with Bonferroni post-test). (C) Mouse primary fibroblasts or MODE-K IEC were incubated with medium or SuperMApo, with or without neutralizing antibodies to TGF-ß, IGF-I or VEGF, or control isotype, and migration was determined using a transwell assay during 24 (h). Data are shown as representative pictures and as pooled data expressed as mean of migrated cells/field of duplicates per group ± s.e.m. from one representative experiment out of three. **p < 0.01, ****p < 0.0001 (one-way ANOVA with tukey’s multiple comparisons post-test).

Fig 4: SCF and IGF-1 accelerate fetal spleen erythropoiesis in vivo.(A) Gating strategy of flow cytometric analysis. CD45-c-Kit-CD71+/-Ter119+ defines terminally-differentiating erythroid cells and mature red blood cells. (B) Graph showing the number of terminally-differentiating erythroid cells and mature red blood cells in mice treated with DMSO, the SCF inhibitor, the IGF-1R inhibitor or both, two hours after injection (n = 3). (C) Relative expression (RQ) of erythroid genes such as Gata1, Klf1 and ß-major globin mRNAs in CD45- cells was examined by real-time PCR. CD45- cells from mice injected with DMSO served as controls (n = 3). Data are means±standard deviation (SD). *P<0.05.

Fig 5: p110 mutations disrupt PI3K signaling and expression of MAPK response genes.(A) Segregation of E14.5 control, p110RBD, and p110CKO lacrimal gland transcriptomes shown by cluster analysis. (B) GO analysis showed that the differentially regulated genes in p110CKO mutants were enriched in PI3K-regulated pathways. ECM, extracellular matrix. (C) GSEA revealed that IGF signaling was disrupted in p110CKO mutants. FDR, false discovery rate. (D) As shown in volcano plot of the control versus the p110CKO transcriptome, Igf1 and Igf2 were among up-regulated genes, and Fgf1, Etv4, Etv5, Dusp6, Six1, and Sox10 were significantly down-regulated. (E) Heatmap of differentially regulated genes implicated in IGF and MAPK pathways. (F to W) RNA in situ hybridization and immunostaining confirmed down-regulation of MAPK response genes in p110RBD and p110CKO mutants.