Fig 2: GluR2 is expressed in EC cell lines and tissues. (A-D) IHC analysis of GluR2 in normal endometrium (N), atypical hyperplasia endometrium (AH), EC and stage I-III EC tissue. The mean level of GluR2 (encoded by GRIA2 gene) was significantly higher in AH than in normal and EC groups, whereas no significant difference was detected in different EC stages. Semiquantitative analysis of IHC staining was practiced among the three groups; scale bar, 50µm. (E, F) The expression of GluR2 at the protein level in EC cell lines was detected by western blotting, and the results were further quantified by densitometry three times. (G) Representative image of neuro-filament light chain (NF-L) in AH tissues (the long and linear fiber stained in green, red arrow); scale bar, 50µm. (H) The number of nerve fibers in normal, AH and EC tissues were counted in 10 random fields/slide and statistically analyzed; scale bar, 50µm. Data are presented as the mean ± SD; *P< 0.05.

Fig 3: Foretinib delays local axon degeneration caused by chemotherapeutic agents or axotomy and pro-Wallerian degeneration signaling. (A) Bright-field micrographs of rat sympathetic neurons exposed to 40 nM of the chemotherapeutic agent vincristine (Vinc) with or without 500 nM foretinib (Foret) for 10 h. The insets show higher magnification views. (B) Ratio of intact neurites as in A. **, P < 0.01; n = 3 independent experiments. (C) Quantitative RT-PCR detected changes in bimEL mRNA levels as in A after 6 h; n = 3 independent experiments. **, P < 0.01. (D) WB of mouse sympathetic neurons treated with vincristine with or without foretinib for 1 h and probed for phosphorylated (p) and total MKK4 or phosphorylated and total JNK. (E) Foretinib delays Wallerian degeneration after axotomy. Compartmented cultures of rat sympathetic neurons where cell bodies and proximal axons were removed 15 min after the addition of 500 nM foretinib to one side compartment and immunostained for ßIII-tubulin (white) 16 h after axotomy. Images show axons in the two side compartments of the same culture. (F) Quantification of the number of beads per 50 µm of neurites in the side compartments of cultures as in E. **, P < 0.01; n = 3 independent experiments. (G–J) WB analysis of isolated axons lysed 15 min (G), 10 h (H), 4 h (I), or 8 h (J) after cell body removal and probed with the indicated antibodies. (H) Neurofilament L (NF-L), aII-spectrin (aIIspec), anti-a-internexin (aintrnx); t0 represents axons lysed immediately after isolation. (K) Axonal ATP levels measured 6 and 9 h after ganglia removal in the absence (control) or presence of foretinib, presented as the percentage of ATP in axons processed immediately after isolation (t0 control). *, P < 0.05; **, P < 0.01; n = 4. (L and N) Foretinib suppresses axon degeneration caused by increases in prodegenerative NMN. Isolated mouse sympathetic neuron axons were cultured in foretinib or the endogenous NMN synthesis inhibitor FK866 in the presence or absence of NMN for 9 h after axotomy. (M) FK866 and foretinib together result in protection of severed axons for up to 72 h. (N) Quantification of the number of beads per 50 µm of isolated neurites as in L. *, P < 0.05; **, P < 0.01; ***, P < 0.005; n = 3 independent experiments. (O) Foretinib protects mitochondria in isolated axons. Intact or axotomized (lesioned) mouse sympathetic neurons treated or not for 9 h with foretinib (bright-field images) and then stained with TMRM to label active mitochondria (fluorescent images). (P) Quantitation of images as in O, presence of live mitochondria per total axons. ***, P < 0.001; n = 3. In F–K, N, and P, “untr” refers to untreated cultures. Bars: (A, inset) 12.5 µm; (A, main, E, L, M, and O) 50 µm. Error bars represent mean ± SEM.

Fig 4: Expression levels of Tace and phosphorylation of ErbB2 in Mtmr2 -/- sciatic nerves and Schwann cell/DRG neuron co-cultures Western blot analysis of Tace using lysates from WT and Tace Fl/Fl;P0-Cre mouse sciatic nerves at P10 (65 µg of total protein lysate loaded). Representative of two independent experiments.Western blot analysis (50 µg of total protein lysate loaded) shows that Tace expression in axons is maximal at P5-P10 and then progressively declines in the adult. Representative of two independent experiments.Western blot analysis of Tace in Mtmr2 -/- sciatic nerves at P5, P10, and P20. Representative of two independent experiments.Western blot analysis of Tace using lysates from Mtmr2 -/- mouse sciatic nerves at 5 months, with quantification, P = 0.10, two-tailed Mann–Whitney U-test. Representative of two independent experiments.Phosphorylation of ErbB2 in Mtmr2 -/- sciatic nerves at P2, P10, and P15. At P2, each lane is a pool of n = 7 animals per genotype, representative of six independent experiments using n = 6 different nerve pools per genotype, and quantification using WT values arbitrary set to 1. *P = 0.0313, Wilcoxon rank-sum test. At P10 and P15, mean values of two samples are shown. At P10, each lane is a pool of n = 3/4 animals per genotype, representative of three independent experiments using n = 6 different pools per genotype. Nf-l, neurofilament light chain; Clx, calnexin.Phosphorylation of ErbB2 in Mtmr2 -/- Schwann cell/DRG neuron co-cultures after 4 days of ascorbic acid treatment. Each lane is a pool from at least 10 coverslips/DRG per genotype. Representative of two independent experiments using n = 4 different pools of coverslips/DRG per genotype, P = 0.20, two-tailed Mann–Whitney U-test.Data information: Results in (D–F) are expressed as mean ± SEM. In (A–D), arrowheads indicate the pro-protein and arrows show the cleaved active form of Tace.Source data are available online for this figure.

Fig 5: Difference of nucleus between synaptic and non-synaptic regions at NMJs. A Whole-mount staining of soleus muscles from 2-month-old C57BL/6 J mice. Note that several nuclei are enriched at R-BTX-labeled synaptic sites (indicated with dotted lines). R-BTX (red). B Quantification of number of nucleus in the synaptic region (SR) or the non-synaptic region (NSR) in A. n = 38 myofibers. Results were from three mice. C Schematic diagram of the separation of SR and NSR. D Immunoblot showing that the expression of NF-L and Myl4 in SR and NSR. Tubulin was used as a loading control. E Heat-map of RNA-seq results showing differentially expressed genes (DEGs) in SR and NSR of soleus muscles. Fold change (FC) > 1.3; q < 0.001. Muscle samples (SR and NSR) from nine mice were randomly divided into three groups, and mRNA from each group of samples was individually extracted and processed for RNA-seq. F Gene Ontology (GO) analysis showing the enriched biological processes of DEGs between SR and NSR. G Verification of DEGs in SR and NSR by real-time PCR analysis. H Fluorescence image showing the different chromatin organization in SR and NSR. The heterochromatin is indicated by anti-H3K9me3 (red), which shows a similar pattern of DAPI staining (blue). BTX-488 (green). I–K Quantification in H. (I). Area of each chromocenter. n = 76 nuclei in NSR and n = 79 in SR. (J). Number of chromocenter. n = 63 nuclei in NSR and n = 65 in SR. (K); total area of chromocenter in each nucleus. n = 51 nuclei in NSR and n = 53 in SR. Data were from three mice. Unless otherwise specified, at least three independent experiments were performed; the mean ± SEM is shown; t-test in B, G, I, J, and K; *p < 0.05, **p < 0.01, and ***p < 0.001