Journal: Molecular Pain

Article Title: MicroRNA-9 regulates mammalian axon regeneration in peripheral nerve injury

doi: 10.1177/1744806917711612

Figure Lengend Snippet: MiR-9 overexpression inhibited axon regeneration in adult sensory neurons in vitro and in vivo. (a) qRT-PCR data indicating miR-9 levels in adult dorsal root ganglions after sciatic nerve injury. Note that miR-9 expression was significantly down-regulated from three to seven days after sciatic nerve axotomy ( n = 3 for each condition). Error bars represent SEM. ** P < 0.01. (b) and (c) Quantification of miR-9 mRNA level at three days after electroporation of miR-9 mimics in vivo and in vitro ( n = 3 for each condition). Error bars represent SEM. ** P < 0.01. (d) Representative images of EGFP-labeled regenerating axons in the whole-mount sciatic nerves. Red arrowheads mark the crush sites. Bar = 500 µm. (e) Scatter plot of average lengths of regenerating sciatic nerve axons ( n = 6 mice for each condition). Error bars represent SEM. ** P < 0.01. (f) Representative images of cultured adult mouse sensory neurons expressing EGFP (green), EGFP + miR-9 mimics (miR-9). All neurons were stained with Tuj1 (red). Scale bar = 100 µm. (g) Quantification of the average length of the longest axons (normalized to the average length of the control axons, n = 3). Error bars represent SEM. ** P < 0.01.

Article Snippet: To transfect RNA oligos into dissociated DRG neurons, the neurons were centrifuged to remove the supernatant and then resuspended in 80–100 μl of Amaxa electroporation buffer (for mouse neuron) with siRNA and miRNAs mimics (0.2 nmol per transfection) and/or the enhanced green fluorescence protein (EGFP) (10 µg per transfection) plasmid.

Techniques: Over Expression, In Vitro, In Vivo, Quantitative RT-PCR, Expressing, Electroporation, Labeling, Cell Culture, Staining

Journal: Molecular Pain

Article Title: MicroRNA-9 regulates mammalian axon regeneration in peripheral nerve injury

doi: 10.1177/1744806917711612

Figure Lengend Snippet: FoxP1 is target gene of miR-9 in adult sensory neurons during axon regeneration. (a) Representative Western blot images of FoxP1 in cultured adult dorsal root ganglion (DRG) neurons three days after transfection of miR-9 mimics. Overexpression of miR-9 leads to decreased FoxP1 protein level. (b) Quantification of FoxP1 level in vitro (normalized to actin, n = 3 for each condition). Error bars represent SEM. ** P < 0.01. (c) Representative Western blot images of FoxP1 in cultured adult DRGs in vivo three days after electroporation of miR-9 mimics. (d) Quantification of FoxP1 level in vivo (normalized to actin, n = 3 for each condition). Error bars represent SEM. ** P < 0.01.

Article Snippet: To transfect RNA oligos into dissociated DRG neurons, the neurons were centrifuged to remove the supernatant and then resuspended in 80–100 μl of Amaxa electroporation buffer (for mouse neuron) with siRNA and miRNAs mimics (0.2 nmol per transfection) and/or the enhanced green fluorescence protein (EGFP) (10 µg per transfection) plasmid.

Techniques: Western Blot, Cell Culture, Transfection, Over Expression, In Vitro, In Vivo, Electroporation

Journal: The Journal of Biological Chemistry

Article Title: Effect of Ser-129 Phosphorylation on Interaction of α-Synuclein with Synaptic and Cellular Membranes *

doi: 10.1074/jbc.M111.253450

Figure Lengend Snippet: Binding of dephosphorylated or phosphomimic α-syn to synaptic membranes. A, cytosol obtained from the brains of transgenic mice expressing the human form of WT, A30P or A53T α-syn was incubated with or without CIP for 1 h at 37 °C. The CIP-treated cytosol was then incubated with α-syn−/− synaptic membranes for 10 min at 37 °C, and the level of membrane-bound α-syn was quantified. B, representative Western blots showing the level of total (T), cytosolic (C), and membrane-bound (M) α-syn and Ser(P)-129 α-syn are shown. C, SHSY5Y cells were transfected with either WT, S129A, or S129D α-syn, and the level of membrane-bound α-syn was quantified 48 h post-transfection. D, representative Western blots showing the level of membrane-bound (M) and cytosolic (C) α-syn and syntaxin in untransfected (UT) and transfected (WT, S129A, and S129D) lysates are shown.

Article Snippet: 4 × 10 6 SHSY5Y cells were resuspended in 100 μl of transfection buffer (Amaxa Biosystems, Gaithersburg, MD).

Techniques: Binding Assay, Transgenic Assay, Expressing, Incubation, Membrane, Western Blot, Transfection

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Enhanced activation of human dendritic cells by silencing SOCS1 and activating TLRs simultaneously

doi: 10.1007/s00262-012-1218-4

Figure Lengend Snippet: Phenotypic analysis of DCs. At 24 h after nucleofection, DCs were harvested and stained with mAbs against DCs surface markers or appropriate isotype control antibody as described in “Materials and methods”. Percentage of positive DCs is indicated. One representative experiment of five is shown

Article Snippet: Nucleofection DCs were harvested at day 6 of culture, washed once in PBS/1%BSA and resuspended in specified nucleofection buffer from Amaxa (Germany) at a final concentration of 2 × 10 7 cells/ml.

Techniques: Staining, Control

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Enhanced activation of human dendritic cells by silencing SOCS1 and activating TLRs simultaneously

doi: 10.1007/s00262-012-1218-4

Figure Lengend Snippet: Th1 cytokines secreted by pshS1NH–DCs were higher than those secreted by non-nucleofected DCs and pNC-GFP-DCs. 1 non-nucleofected DCs; 2 pNC-GFP-DCs; 3 pshS1NH–DCs. At 48 h after nucleofection, supernatants were collected for TNF-α, IL-6, IL-1β and IL-12p70 ELISA analyses. The pshS1NH–DCs secreted significantly higher level of Th1 cytokines than the pNC-GFP-DCs and non-nucleofected DCs (P < 0.05). Data shown are the means ± SEM and representative of six independent experiments. The significant differences between pshS1NH–DCs and control were determined by ANOVA

Article Snippet: Nucleofection DCs were harvested at day 6 of culture, washed once in PBS/1%BSA and resuspended in specified nucleofection buffer from Amaxa (Germany) at a final concentration of 2 × 10 7 cells/ml.

Techniques: Enzyme-linked Immunosorbent Assay, Control