Additional file 1: Figure S1. of Subventricular zone neural progenitors reverse TNF-alpha effects in cortical neurons

AM251 treatment has no effect on synaptic activity, cell viability and CB1 receptor expression. A Representative electrophysiological traces and group data of average mEPSC amplitude and frequency of control (n = 10) and AM 251 treated (n = 9) cells (scale bar: 20 pA, 200 ms). B Fluorescence images of Hoechst and propidium iodide double staining in cortical neurons and summary bar graph showing lack of cytotoxic effects after AM251 treatment (scale bar: 40 μm). C, D Confocal microscopy images and summary bar graph showing expression of CB1 receptors in control and AM251 treated cultured neurons (scale bar: 20 μm). All data are expressed as mean ± standard error of the mean. CB1 type 1 cannabinoid receptor, mEPSC miniature excitatory post-synaptic current. (PPT 1143 kb

Additional file 1: Figure S1. of IL4 induces IL6-producing M2 macrophages associated to inhibition of neuroinflammation in vitro and in vivo

Gene expression of typical M1 and M2 markers in peritoneal macrophages stimulated in vitro. (A–D) real-time RT-PCR for iNOS, Ym1, CCI17, and iL6, in non stimulated (NS), and in macrophages stimulated with different doses of iFn-γ (m1) and iL4 (M2). Gapdh has been used as a housekeeping gene. Data are shown as fold induction (Fi ± standard deviation) over NS. (TIF 42402 kb

Additional file 6: of Selective killing of spinal cord neural stem cells impairs locomotor recovery in a mouse model of spinal cord injury

Movie S2 Video of Catwalk gait analysis of a representative GCV-NestinTK mouse. NestinTK mice were treated with GCV, subjected to SCI and followed for locomotor recovery by BMS score when behavioral amelioration reached a plateau value, mice were subjected to Catwalk gait analysis. The video shows recording of a representative GCV-NestinTK animal (BMS score = 1). Free ambulation along an illuminated glass plate in a darkened room has been recorded for 3.22 s. Video has been captured at day 18 after SCI. (AVI 104621 kb

Additional file 8: of Selective killing of spinal cord neural stem cells impairs locomotor recovery in a mouse model of spinal cord injury

Figure S6. Upregulation of inflammatory cues in GCV-NestinTK mice. Real-time PCR analysis of pro-inflammatory genes (A–D) in T11–T13 spinal cord tissues at different time points after the injury induction. GCV-NestinTK mice (red bars) have a increased expression of pro-inflammatory genes after injury compared with control mice (blue bars). Values indicate mean fold changes ± S.E.M (n = 3–6 for each group). Comparisons were done using the t Student test: TNFα:* p = 0.021; IL-1β:* p = 0.046; Vegfa: p = 0.008. (TIFF 7997 kb

Additional file 5: of Selective killing of spinal cord neural stem cells impairs locomotor recovery in a mouse model of spinal cord injury

Movie S1 Video of Catwalk gait analysis of a representative GCV- WT mouse. WT mice were treated with GCV, subjected to SCI and followed for locomotor recovery by BMS score when behavioral amelioration reached a plateau value mice, were subjected to Catwalk gait analysis. The video shows recording of a representative GCV-WT animal (BMS score = 3). Free ambulation along an illuminated glass plate in a darkened room has been recoded for 4.30 s. Video has been captured at day 18 after SCI. (AVI 139494 kb

Additional file 7: of Selective killing of spinal cord neural stem cells impairs locomotor recovery in a mouse model of spinal cord injury

Figure S5. macrophages/macrophages activation affects both GCV-WT and GCV-NestinTK mice. (A) Histological analysis of a SC region located 3 mm far from the injury site from a GCV-WT mice (18 days post injury). Microglia/macrophages are labeled for Iba1 and F4/80. Arrowhead indicates cells that are shown at high magnification in panel A’. Panel B shows the site of the injury in the SC of GCV-WT mouse. Arrowhead indicates cells that are shown at high magnification in panel B′. A representative section of the SC located 3 mm far from the site of the injury from a GCV-NestinTK mouse (18 days post injury) is shown in panel C. Arrowhead indicates cells that are shown at high magnification in panel C′. Panel D shows the site of the injury while the arrowhead indicates cells that are shown at high magnification in D’ (n = 3 for each group). Scale bar 50 μm (TIFF 4124 kb

Additional file 3: of Selective killing of spinal cord neural stem cells impairs locomotor recovery in a mouse model of spinal cord injury

Figure S3. GCV treatment ablates proliferating SC-eNSCs in NestinTK mice. Panels A and B show representative confocal images of VIM (red) and Brdu (green) in the ependymal layer of GCV-WT (A) and GCV-NestinTK (B) mice (n = 3 for each group). Mice were sacrificed at the end of the GCV treatment. Quantifications (means ± S.E.M.) are shown in panel C. Two-way ANOVA followed by Bonferroni’s multiple Comparison test has been used to analyze data. ** p = 0.011 and p = 0.045 in thoracic and lumbar segments, respectively. Scale bar 20 μm. (TIFF 2754 kb

Additional file 2: of Selective killing of spinal cord neural stem cells impairs locomotor recovery in a mouse model of spinal cord injury

Figure S2. Sorted GFP+ cells from Nestin floxGFPflox-TK mice give rise to neurospheres. Panel A and B show the gating strategy for sorting GFP+ cells from SCs bulk cultures obtained from Nestin floxGFPflox-TK mice. WT litters (A) were used to set up the gating strategy that we used to sort GFP+ cells (B). GFP+ cells were plated at the density of 8000 cells/cm2 and daily examined for the presence of neurospheres. Small spheres were observed after 3 days (C), while spheres with diameters larger than 100 μm were easily observed after 7 days (D). Scale bar 50 μm (n = 3 independent preparations). (TIFF 9717 kb

FoxP3⁺CD39⁺ Treg cells are increased during acute MS.

<p>Scatter plots from three representative subjects, one healthy donor (HC; <b>A</b>), a stable MS patients (<b>B</b>), and a patient experiencing a clinical re-exacerbation of MS (<b>C</b>) are shown, indicating that the FoxP3/CD39 double positive T cell population (<b>A</b>, right panel) in the CD25^high gate dramatically decreases during stable MS (<b>B</b>, right panel) and is restored during an acute attack (<b>C</b>, right panel).</p

CD4⁺CD25^highCD39⁺ T cells express increased levels of Treg markers.

<p>FoxP3 (<b>A</b>), CTLA4 (<b>B</b>) and GITR (<b>C</b>) expression, as measured by flow cytometry, are increased in CD4⁺CD39⁺, as compared to CD4⁺CD39⁻ T cells, especially in the CD25^high compartment.</p