Fig 1: Neuron-glial antigen 2 (NG2) expression in injured spinal cords. (A) Representative image of NG2 expression (red) in the marginal regions of the injured spinal cord at 2 week post-injury with yellow dashed line demarcating lesion edge and asterisk the lesion core. (B) NG2 (green) colocalization with F4/80 (red) positive cells within the marginal region of the injured cord at 1 week post-injury. (C–E) Representative image showing colocalization between RFP+ bone marrow-derived cells (BMDCs) and NG2 (purple) within the marginal region at 1 week (C) and 2 week (E) post-injury in a cord from an RFP bone marrow (BM) chimeric, with yellow arrows highlighting two NG2+ BMDCs. (D) Quantification of RFP+ BMDCs with or without NG2 colocalization within the marginal region 1 week post-injury; data are shown as means ± SD (n = 4), with at least three images quantified per mouse from four separate mice. *P = 0.05. (F) Representative image showing colocalization between GFP+ BMDCs and NG2 (red) within the marginal region in a 2 week post-injury cord from a GFP bone marrow chimeric mouse; data are shown as means ± SD (n = 4), with at least three images quantified per mouse from four separate mice. *P = 0.05. (G) Representative image showing colocalization between CX3CR1+++ (green) and NG2 (red) within the marginal region at 10 days post-injury cord from CX3CR1GFP/+ mouse. The yellow arrows highlighting two microglia expressing NG2. (H) Quantification of CX3CR1+++ microglia with and without NG2 colocalization within the marginal region; data are shown as means ± SD (n = 3), with at least three images quantified per mouse from three separate mice. *P = 0.05.
Fig 2: NG2 expression at levels of the mRNA, intracellular protein, and secreted protein in BMDMF treated with myelin debris. (A) NG2 mRNA expression in BMDMF treated with myelin debris for 7 days. (B) NG2 protein expression in BMDMF treated with myelin debris for 7 days and 14 days assessed by WB images. Corresponding quantification of protein levels was determined by densitometry analysis relative to a-tubulin. The immunoblots were performed twice with similar results. C: control; T: myelin debris treatment. (C) NG2 protein expression in microglia with myelin debris for indicated time points assessed by WB images. Corresponding quantification of protein levels was determined by densitometry analysis relative to a-tubulin. The immunoblots were performed four times with similar results. C: control; T: myelin debris treatment. (D) Chondroitin sulfate proteoglycan 4 (CSPG4) in the supernatant of BMDMF treated with myelin debris for 7 days and 14 days detected by Enzyme-linked immunosorbent assay (ELISA). Data for all quantifications are shown as means ± SD of three separate BMDMF isolations from three separate mice (n = 3). **P = 0.01.
Fig 3: Graphical representation of the induction and consequences associated with NG2 expression in BMDMF. BMDMF upon treatment with myelin debris demonstrate an increase in expression and secretion of NG2/CSPG4 in a small percentage of their cell population. These NG2 positive cells demonstrate increased proliferation and possess a lower phagocytic capacity than NG2 negative BMDMF.
Fig 4: Alteration of proliferation and phagocytic capacity in NG2+ BMDMF. (A) Representative image of NG2 expression (red) and proliferation marker BrdU (green) in naïve BMDMF (absence of myelin debris; left) and BMDMF treated myelin debris for 14 days (right). (B) Representative image of EdU+ (red) with NG2+ (purple) BMDMF treated myelin debris for 14 days; inset showing detailed immunostaining of a single EdU+/NG2+ BMDMF. (C) Percentage of EdU+ in NG2+ BMDMF and NG2- BMDMF. (D) Representative image of BMDMF phagocytosis of myelin debris in NG2+ BMDMF and NG2- BMDMF. Engulfed myelin debris was determined by detection of intracellular myelin basic protein (MBP; red) puncta; insert showing detailed immunostainings of NG2+ BMDMF and NG2- BMDMF containing MBP puncta. (E) The corresponding percentage of NG2+ BMDMF and NG2- BMDMF phagocytosis of myelin debris. (F) Representative image of BMDMF phagocytosis of latex beads (red) in NG2+ BMDMF and NG2- BMDMF; insert showing detailed immunostaining of NG2+ BMDMF and NG2- BMDMF containing latex beads. (G) The corresponding percentage of latex beads engulfment in NG2+ BMDMF and NG2- BMDMF. (H) The number of engulfed beads in both NG2+ BMDMF and NG2- BMDMF. Data are shown as means ± SD of three separate BMDMF isolations from three separate mice (n = 3). *P = 0.05 and **P = 0.01.
Fig 5: Myelin debris engulfment stimulates NG2 expression in BMDMF. (A) Percentage of NG2 expressing BMDMF after treatment with myelin debris or TGF-ß for 14 days. (B) Representative images of NG2 expression (red) in BMDMF (F4/80, purple) cultured and treated with myelin debris for 14 days (left) or cultured for 14 days and then treated with myelin debris for 3 h (right). (C) The proportion of NG2 expressing BMDMF treated with myelin debris for 7 days and 14 days, respectively. Data for all quantifications are shown as means ± SD of three separate BMDMF isolations from three separate mice (n = 3). **P = 0.01 and ****P = 0.0001. (D) NG2 protein in myelin debris and mouse primary pericytes detected by Western blot (WB) images. Corresponding quantification of protein levels was determined by densitometry analysis relative to a-tubulin.
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