Journal: Stem cells (Dayton, Ohio)

Article Title: Mifepristone-inducible caspase-1 expression in mouse embryonic stem cells eliminates tumor formation but spares differentiated cells in vitro and in vivo.

doi: 10.1002/stem.1000

Figure Lengend Snippet: Figure 4. Survival of differentiated dopamine neurons derived from caspase-1-ESCs treated with mifepristone in vitro. (A): Mifepristone had no effect on differentiated caspase-1-ESCs after 6 days treatment. Scale bar ¼ 500 lm. (B): Mifepristone had no effect on differentiated WT and cas- pase-1-ESCs. (C): Fluorescence-activated cell sorting (FACS) results showed that mifepristone did not affect differentiated WT or caspase-1-ESCs; (C0) was the quantitative analysis from (C) (n ¼ 3). (D): Mifepristone did not induce DNA cleavage in differentiated caspase-1-ESCs. Scale bar ¼ 10 lm. (E): The protein levels of caspase-1, p20 and p10 expression in differentiated caspase-1-ESCs showed increase after mifepristone treatment; (E0) was the quantitative analysis from (E) (n ¼ 3). *, p < .05 (compared with caspase-1-ES vehicle). (F): MAP2þ/THþ caspase-1-ESCs survived in the presence of mifepristone. Scale bar ¼ 50 lm. (G): Mifepristone treatment did not affect THþ or MAP2þ colony proportion. Colony counts were per- formed in triplicate and at least 200 colonies were counted each time. Abbreviations: ESC, embryonic stem cell; MAP2, microtubule-associated pro- tein 2; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PI, propidium iodide; TH, tyrosine hydroxylase; WT, wild type

Article Snippet: After fixing the cells and sections, primary antibodies were applied as follows: mouse anti–microtubule-associated protein 2 (anti-MAP2) antibody (1:2,000, M4403, Sigma), rabbit anti-tyrosine hydroxylase (TH) antibody (1:500, AB152, Chemicon), mouse anti–sex-determining region Y protein (anti-SRY) antibody (1:200, ab22166, Abcam, UK, www.abcam.com), rabbit anti-Oct-4 antibody (1:200, 2750, Cell Signaling), mouse anti-nestin antibody (1:200, MAB353, Chemicon), and goat anti-MAP2 antibody (1:20, sc-5359, Santa Cruz).

Techniques: Derivative Assay, In Vitro, Fluorescence, FACS, Expressing

Journal: Stem cells (Dayton, Ohio)

Article Title: Mifepristone-inducible caspase-1 expression in mouse embryonic stem cells eliminates tumor formation but spares differentiated cells in vitro and in vivo.

doi: 10.1002/stem.1000

Figure Lengend Snippet: Figure 5. Ablation of tumors and survival of dopamine neurons derived from caspase-1-ESCs in brain after mifepristone treatment. (A): Undifferenti- ated WT ESCs and caspase-1-ESCs were Oct-4þ. Most differentiated WT and caspase-1-ESCs were nestinþ. Scale bar ¼ 10 lm. (B): The mifepristone treatment increased the life span of mice transplanted with undifferentiated caspase-1-ESCs (n ¼ 6). (C): WT ESCs formed tumors in the absence or pres- ence of mifepristone, while caspase-1-ESCs formed tumors only in the absence of mifepristone. (D): The hematoxylin and eosin staining verified that the tumors were derived from ESCs. Scale bar ¼ 50 lm. (E): Dopamine neurons derived from caspase-1-ESCs appeared in both vehicle- and mifepristone- treated mice brains. The arrows indicated the SRYþ/THþ/MAP2þ cells. (F): Quantitative analysis of SRYþ/THþ/MAP2þ cells in the brain showed that mifepristone did not affect dopamine neurons derived from caspase-1-ESCs (n ¼ 4). Scale bar ¼ 10 lm. Abbreviations: ESC, embryonic stem cell; MAP2, microtubule-associated protein 2; SRY, sex-determining region Y protein; TH, tyrosine hydroxylase; WT, wild type.

Article Snippet: After fixing the cells and sections, primary antibodies were applied as follows: mouse anti–microtubule-associated protein 2 (anti-MAP2) antibody (1:2,000, M4403, Sigma), rabbit anti-tyrosine hydroxylase (TH) antibody (1:500, AB152, Chemicon), mouse anti–sex-determining region Y protein (anti-SRY) antibody (1:200, ab22166, Abcam, UK, www.abcam.com), rabbit anti-Oct-4 antibody (1:200, 2750, Cell Signaling), mouse anti-nestin antibody (1:200, MAB353, Chemicon), and goat anti-MAP2 antibody (1:20, sc-5359, Santa Cruz).

Techniques: Derivative Assay, Staining

Journal: Neuroscience research

Article Title: Parallel high throughput neuronal toxicity assays demonstrate uncoupling between loss of mitochondrial membrane potential and neuronal damage in a model of HIV-induced neurodegeneration

doi: 10.1016/j.neures.2011.01.013

Figure Lengend Snippet: (a) MAP2 RMF in the linear range of fluorescence is maximal (Brewer, 1995) compared with no primary control background when 0.5μg of Fluorocillin substrate is incubated for 60 min. (b) Background-subtracted MAP2 RMF values increase linearly with increased plating density of primary rat cortical neurons sevaluated after 3 days in culture. n=2-3 wells (a), n=14-16 wells (b) one-way ANOVA, Newman-Keuls, p<0.05 for all differences in cell densities ≥10×103; in some cases the SEM was smaller than the plotting symbol.

Article Snippet: After blocking for 1 h with 5% normal goat serum in phosphate buffered saline (PBS), plates were incubated with mouse anti-MAP2 antibody (SMI-52, 1:1000, Covance, Berkley, CA) diluted in Normal Antibody Diluent (ScyTek Labs, Logan, UT, USA) overnight at 4°C, followed by washing with PBS with 0.1% Tween-20 (PBS-T).

Techniques: Fluorescence, Control, Incubation

Journal: Neuroscience research

Article Title: Parallel high throughput neuronal toxicity assays demonstrate uncoupling between loss of mitochondrial membrane potential and neuronal damage in a model of HIV-induced neurodegeneration

doi: 10.1016/j.neures.2011.01.013

Figure Lengend Snippet: (a) Cultures were labeled for MAP2 (red) and DAPI (blue) and imaged by immunofluorescent laser confocal microscopy at various timepoints (2, 4, and 20 hr) following the application of HIV- or Mock-MDM (1:10 dilutions). In HIV-MDM-treated cultures, dendritic beading is evident by 2hr, and widespread loss of MAP2-positive neurons and dendrites, as well as condensed nuclei, is pronounced by 20h. (b) 96-well plate cortical cultures were treated with serial dilutions of HIV-MDM for 40 hr and were assayed for changes in MAP2-positive neurons by MAP2 CB-ELISA (left) or by hand-counting MAP2-positive cells (right). Each dilution of HIV-MDM resulted in a significant decrease in % of MAP2 RMF (left) or % of neuron number (right) when compared with untreated. Each dilution of HIV-MDM was also significantly different from all other HIV-MDM treatments evaluated by the same assay (p<0.001 one-way ANOVA, Newman-Keuls). For any particular dilution of HIV-MDM, there was no statistical difference in the % changes from untreated obtained from the MAP-CB ELISA (MAP2 RMF values) compared with the % change from untreated obtained from traditional hand-counting assay (number of MAP-2 positive cells) in the same plate (p<0.05, one-way ANOVA, Bonferroni), n=6-12 wells. (c) 96-well plate cortical cultures were treated with serial dilutions of glutamate for 24 hr. Values are shown normalized to vehicle control. Each increase concentration of glutamate resulted in a statistically significant (p<0.001, one-way ANOVA, Newman-Keuls) observed increase in MAP2 RMF compared with all other dilutions, with two exceptions: for 1μM glutamate compared with vehicle p<0.05, and 10μM compared with 5μM is not statistically different.

Article Snippet: After blocking for 1 h with 5% normal goat serum in phosphate buffered saline (PBS), plates were incubated with mouse anti-MAP2 antibody (SMI-52, 1:1000, Covance, Berkley, CA) diluted in Normal Antibody Diluent (ScyTek Labs, Logan, UT, USA) overnight at 4°C, followed by washing with PBS with 0.1% Tween-20 (PBS-T).

Techniques: Labeling, Confocal Microscopy, Enzyme-linked Immunosorbent Assay, Control, Concentration Assay

Journal: Neuroscience research

Article Title: Parallel high throughput neuronal toxicity assays demonstrate uncoupling between loss of mitochondrial membrane potential and neuronal damage in a model of HIV-induced neurodegeneration

doi: 10.1016/j.neures.2011.01.013

Figure Lengend Snippet: Loss of MAP2 RMF as measured by MAP2 CB-ELISA in 2-week old cortical cultures either untreated or following treatment with 1:10 or 1:40 dilutions of HIV-MDM at 3, 6, 12, 24, 36, and 60 hr. By 12 hr, both dilutions of HIV-MDM showed significant loss of MAP2 RMF compared with untreated cultures. MAP2 RMF loss following treatment with a 1:40 dilution of HIV-MDM was significantly reversed at 60 hr compared with 36 hr. n=12-16 wells; one-way ANOVA, Newman-Keuls, * p<0.01 compared with 36 hr; in some cases the SEM was smaller than the plotting symbol.

Article Snippet: After blocking for 1 h with 5% normal goat serum in phosphate buffered saline (PBS), plates were incubated with mouse anti-MAP2 antibody (SMI-52, 1:1000, Covance, Berkley, CA) diluted in Normal Antibody Diluent (ScyTek Labs, Logan, UT, USA) overnight at 4°C, followed by washing with PBS with 0.1% Tween-20 (PBS-T).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Neuroscience research

Article Title: Parallel high throughput neuronal toxicity assays demonstrate uncoupling between loss of mitochondrial membrane potential and neuronal damage in a model of HIV-induced neurodegeneration

doi: 10.1016/j.neures.2011.01.013

Figure Lengend Snippet: (a) 5-week-old cultures were left unexposed or exposed for 20 hr to 1:40 or 1:10 dilutions of HIV- or Mock-MDM with or without treatment with 20μM qVD-OPH, 10μM MDL28170, or 10μM MK801. There were no differences between any conditions in cultures exposed to Mock-MDM. At both dilutions of HIV-MDM, MDL28170 and MK801 significantly reduced the loss of MAP2 RMF. Cultures treated with qVD-OPH did not show statistical differences between treatments. ^p<0.05, *p<0.001 compared with vehicle-treated cultures receiving matching dilution of HIV-MDM (1:10 – black bars; 1:40 – hatched bars), or the vehicle-treated control cultures not receiving HIV-MDM (white bar) compared with vehicle-treated cultures receiving either 1:10 or 1:40 dilutions of HIV-MDM, one-way ANOVA, Newman-Keuls, n=13-30 wells. (b) MAP2 RMF loss in cultures exposed to a 1:10 dilution of HIV-MDM for 24 hr was not reduced when treated with various concentrations of qVD-OPH compared with vehicle-treated (0.5% DMSO) cultures. (c) In a separate experiment, cultures receiving a 1:10 dilution of HIV-MDM for 25 hr had reduced loss of MAP2 RMF when they were treated with 1, 10, and 100 μM MDL28170 compared with vehicle (1% DMSO) treated cultures. For b and c, v=vehicle; ^p<0.05, *p<0.001 compared with vehicle in HIV-MDM-treated cultures, one-way ANOVA, Dunnett’s, n=6-24 wells. All values are presented as a percentage of vehicle-treated control cultures (dash line, open circle). Maximum MAP2 RMF loss is indicated with a dashed line with closed circle. In some cases the SEM was smaller than the plotting symbol.

Article Snippet: After blocking for 1 h with 5% normal goat serum in phosphate buffered saline (PBS), plates were incubated with mouse anti-MAP2 antibody (SMI-52, 1:1000, Covance, Berkley, CA) diluted in Normal Antibody Diluent (ScyTek Labs, Logan, UT, USA) overnight at 4°C, followed by washing with PBS with 0.1% Tween-20 (PBS-T).

Techniques: Control

Journal: Neuroscience research

Article Title: Parallel high throughput neuronal toxicity assays demonstrate uncoupling between loss of mitochondrial membrane potential and neuronal damage in a model of HIV-induced neurodegeneration

doi: 10.1016/j.neures.2011.01.013

Figure Lengend Snippet: Cortical cultures exposed to a 1:20 dilution of HIV-MDM showed decreased TMRM fluorescence (open symbols and dotted lines) by 15 min and decreased MAP2 RMF by 90 min (closed symbols and solid lines). Cccp+oligomycin was used to uncouple mitochondrial electron chain transport and thoroughly depolarize cultures in order to assess background TMRM fluorescence (2.34±0.90) and is indicated by a dotted line. For visual purposes, untreated control cultures are placed near the 0 min time point at slightly offset (−0.8, 0, and 0.8) x-axis positions in order to avoid symbol overlap. ^p<0.05, *p<0.001 compared with untreated, one-way ANOVA, Dunnett’s; †p<0.01, #p<0.001 compared with Mock-MDM at the same time point, unpaired two-tailed t-test, n=6-12 wells; in some cases the SEM was smaller than the plotting symbol.

Article Snippet: After blocking for 1 h with 5% normal goat serum in phosphate buffered saline (PBS), plates were incubated with mouse anti-MAP2 antibody (SMI-52, 1:1000, Covance, Berkley, CA) diluted in Normal Antibody Diluent (ScyTek Labs, Logan, UT, USA) overnight at 4°C, followed by washing with PBS with 0.1% Tween-20 (PBS-T).

Techniques: Fluorescence, Control, Two Tailed Test

Journal: Neuroscience research

Article Title: Parallel high throughput neuronal toxicity assays demonstrate uncoupling between loss of mitochondrial membrane potential and neuronal damage in a model of HIV-induced neurodegeneration

doi: 10.1016/j.neures.2011.01.013

Figure Lengend Snippet: Two-week-old cortical cultures were exposed to a 1:20 dilution of HIV- or Mock-MDM. a) Neuronal damage, as determined by loss of MAP2 RMF by the MAP2 CB-ELISA and by automated image analysis expressed as integrated pixel intensity of MAP2, is significant by 4 hr and is prevented by MDL28170. In addition, exclusion of PI by cells in the cultures is decreased at 4 hr and is also prevented by MDL28170. b) Wells used in the MAP2 assay were reused for immunofluorescent imaging. Images in the left column show MAP2 (green), GFAP (red), and DAPI (blue). Images on the right column show PI labeling (red) in the same field. PI was added prior to fixation of the cultures in order to label only cells with compromised membranes. Little damage is seen by 1 hr (not shown), but by 4 hr loss of MAP2 and increased PI labeling are evident. c) MAP2 loss (solid symbols, solid lines) is detectable prior to loss of PI-exclusion (open symbols, dotted lines) following treatment with HIV-MDM. d) MDL28170 fails to prevent loss of TMRM fluorescence following HIV- or Mock-MDM treatment at 1 or 4 hr. Cccp+oligomycin was used to depolarize cultures in order to assess background TMRM fluorescence (4.43±0.82) and is indicated by a dotted line. ^p<0.05, †p<0.01, *p<0.001 compared with assay specific b) HIV-MDM treated or c) untreated cultures, one-way ANOVA, Newman-Keuls, n=4-16 wells.

Article Snippet: After blocking for 1 h with 5% normal goat serum in phosphate buffered saline (PBS), plates were incubated with mouse anti-MAP2 antibody (SMI-52, 1:1000, Covance, Berkley, CA) diluted in Normal Antibody Diluent (ScyTek Labs, Logan, UT, USA) overnight at 4°C, followed by washing with PBS with 0.1% Tween-20 (PBS-T).

Techniques: Enzyme-linked Immunosorbent Assay, Imaging, Labeling, Fluorescence

Journal: Journal of Neuropathology & Experimental Neurology

Article Title: Expression of the Interleukin 6 System in Cortical Lesions From Patients With Tuberous Sclerosis Complex and Focal Cortical Dysplasia Type IIb

doi: 10.1097/nen.0b013e3181eaeae5

Figure Lengend Snippet: FIGURE 2. Cell-specific distribution of IL-6 in apparently normal CTX, TSC tubers and FCDIIb lesions. (A, B) Hematoxylin and eosin (H&E) staining. Representative photomicrographs of TSC (A) and FCDIIb (B) showing areas of cortical disorganization containing different cell types, including DNs (arrowheads) with high Nissl substance staining and diverse directionality, TS cells with eosinophilic cytoplasm (arrows in A), and BCs with opalescent cytoplasm and eccentric nuclei (arrows in B). (C, D) Weak to moderate IL-6 IR in neurons (arrows in C), endothelial cells (double arrows in D), and glia-like cells (arrowheads in C and D) in neocortex and white matter (WM). (EYG) Strong IL-6 IR in TS cells (insert, E, arrow) and in DNs of different size and shape (arrowheads in EYG). Merged images show colocalization of IL-6 (green) with NeuN (red) (insert, F), and colocalization of IL-6 (green) with MAP2 (red) (insert, G) in DNs. (H) Representative confocal image showing no IL-6 IR (green) colocalization with GFAP (red). (I and J) Interleukin 6 IR in FCDIIb. Weak (double arrows in I) to moderate (arrows and in I insert) IL-6 IR in BCs; strong IL-6 IR in DNs (arrowhead, I) and in GNs with central nuclei (J). Merged images show colocalization of IL-6 (green) with NeuN (red) in GNs (insert, J) and of IL-6 (green) with NF200 (red) in DNs (K). (L) Confocal image shows that no IL-6 IR DNs (arrowhead) or GNs (double arrowheads) are colocalized with GFAP (red, arrows). Merged image show most cells with IL-6 IR (green, BCs) colabeled with GFAP (red) (insert in L). Sections are counterstained with hematoxylin (CYG, I, J) or Hoechst 33258 (inserts in G, K, L). Bars = (AYE, H, L) 50 Km; (F, G, J, K) 10 Km; (I) 25 Km.

Article Snippet: Unauthorized reproduction of this article is prohibited. and antiYglial fibrillary acidic protein (GFAP, mouse monoclonal, 1:1000; Sigma), ii) antiYIL-6 (1:100) or antiYIL-6R (rabbit polyclonal, 1:100) and anti-NeuN (mouse monoclonal, 1:500; Chemicon), iii) antiYIL-6 (1:100) and anti-microtubuleassociated protein 2 (MAP2) (mouse monoclonal, 1:100; Boster), and iv) antiYIL-6 (rabbit polyclonal, 1:100) or antiYIL-6R (rabbit polyclonal, 1:100) and anti-NF200 (mouse monoclonal, 1:100; Boster) in 0.01 M PBS (pH 7.4) containing 1% bovine serum albumin overnight at 4-C. After 3 rinses, the sections were incubated with a mixture of goat anti-rabbit immunoglobulin class GYfluorescein isothiocyanate (1:200; Zhongshan Goldenbridge Biotechnology Co, Beijing, China) and goat antimouse immunoglobulin class GYtetramethyl rhodamine isothiocyanate (1:200; Upstate Biotechnology, Lake Placid, NY) for 1 hour at 37-C. Counterstaining of cell nuclei was performed by incubating the sections with Hoechst 33258 (0.1 Kg/mL; Sigma).

Techniques: Staining

Journal: Journal of Neuropathology & Experimental Neurology

Article Title: Expression of the Interleukin 6 System in Cortical Lesions From Patients With Tuberous Sclerosis Complex and Focal Cortical Dysplasia Type IIb

doi: 10.1097/nen.0b013e3181eaeae5

Figure Lengend Snippet: FIGURE 3. Cell-specific distribution of IL-6Rs in apparently normal CTX, TSC tubers, and FCDIIb cortical lesions. (A) No IL-6R IR is seen in neuronal or glial cells in control CTX. (BYH) Tuberous sclerosis complex. Strong IL-6R IR in DNs of different sizes and shapes in the neocortex and white matter (WM) (arrowheads in BYD) and in TSC cells (insert in D, arrow). Merged images show colocalization of IL-6R (green) with MAP2 (red) (insert in B), of IL-6R (green) with NeuN (red) (insert in C), and of IL-6R (green) with NF200 (red) (EYG) in DNs (arrowheads) and TS cells (arrows), respectively. (H) Representative confocal image showing that no IL-6R+ (green) DNs (arrowheads) or TS cells (arrows) are colocalized with GFAP (red). Scattered GFAP+ reactive astrocytes are colabeled with IL-6R (insert in H, indicated by double arrows). (IYL) Interleukin 6 receptor in FCDIIb. Moderate to strong IL-6R IR in BCs (I, arrows and double arrows). Interleukin 6 receptorYnegative BCs are indicated by an asterisk (J). Strong IL-6R IR was also observed in GNs (K) and DNs (L). Merged images show colocalization of IL-6R (green) with NeuN (red) in GNs (arrowhead) and BCs (arrow) (insert in I) and colocalization of IL-6R (green) with NeuN (red) in DNs (insert in L), respectively. (M) Representative confocal image showing that no IL-6R-positive (green) GNs or DNs (arrowheads) are colocalized with GFAP (red). Merged image showing that most IL-6R+ (green) BCs colabeled with GFAP (red) (arrows in M). Interleukin 6 receptorYnegative BCs are indicated by double arrows. Sections are counterstained with hematoxylin (BYD, IYL) and Hoechst 33258 (insert in B, C; G, H, M). Scale bar = (A) 100 Km; (B, EYG) 30 Km; (C, D, J, L) 20 Km; (H, I, M) 50 Km; (K) 15 Km.

Article Snippet: Unauthorized reproduction of this article is prohibited. and antiYglial fibrillary acidic protein (GFAP, mouse monoclonal, 1:1000; Sigma), ii) antiYIL-6 (1:100) or antiYIL-6R (rabbit polyclonal, 1:100) and anti-NeuN (mouse monoclonal, 1:500; Chemicon), iii) antiYIL-6 (1:100) and anti-microtubuleassociated protein 2 (MAP2) (mouse monoclonal, 1:100; Boster), and iv) antiYIL-6 (rabbit polyclonal, 1:100) or antiYIL-6R (rabbit polyclonal, 1:100) and anti-NF200 (mouse monoclonal, 1:100; Boster) in 0.01 M PBS (pH 7.4) containing 1% bovine serum albumin overnight at 4-C. After 3 rinses, the sections were incubated with a mixture of goat anti-rabbit immunoglobulin class GYfluorescein isothiocyanate (1:200; Zhongshan Goldenbridge Biotechnology Co, Beijing, China) and goat antimouse immunoglobulin class GYtetramethyl rhodamine isothiocyanate (1:200; Upstate Biotechnology, Lake Placid, NY) for 1 hour at 37-C. Counterstaining of cell nuclei was performed by incubating the sections with Hoechst 33258 (0.1 Kg/mL; Sigma).

Techniques: Control

Journal: Cerebral Cortex (New York, NY)

Article Title: Cortical Neuron Migration and Dendrite Morphology are Regulated by Carboxypeptidase E

doi: 10.1093/cercor/bhy155

Figure Lengend Snippet: Knockdown of CPE decreases dendritic branching in cortical neurons in vivo and in cultured hippocampal neurons in vitro. (A) Mice at E14.5 were electroporated in utero with constructs as indicated, and neurons within cortical layer II/III were analyzed at P7. Representative tracings of dendrites are shown for each condition. Scale bar = 50 μm. (B, C) Quantitation of the number of dendrites (B) and total dendrite length (C) of neurons under each condition. Error bars indicate ± SEM. n (neurons) = 26, CTL shRNA; n = 29, CPE shRNA; n = 50, CPE shRNA+rescue. *P < 0.05, **P < 0.01 as determined by one-way ANOVA followed by Dunnett’s multiple comparisons test. (D) Hippocampal neurons were cotransfected with pCAG-mOrange and negative control siRNA (CTL siRNA) or CPE siRNA as indicated at DIV 7. Neurons were fixed and immunostained for GFP and MAP2 at DIV 10. Neurons positive for both GFP and mOrange were assessed for Sholl analysis. Representative mOrange fluorescent images of neurons are shown as inverted black images. Scale bar = 50 μm. (E) Sholl analysis of neurons transfected with the indicated siRNA. Gray line indicates P value is at least less than 0.001 as determined by 2-way ANOVA followed by Sidak’s multiple comparisons test. (F) Sholl analysis within 60 μm from soma. n (neurons) = 84, CTL siRNA; n = 95, CPE siRNA. *P < 0.05, **P < 0.01 as determined by 2-way ANOVA followed by Sidak’s multiple comparisons test. (G) Representative images showing CPE siRNA knockdown efficiency in transfected neurons. Hippocampal neurons were cotransfected with pCAG-mOrange (indicated by arrows) and either CTL siRNA or CPE siRNA at DIV 7, fixed at DIV 10, and immunostained for CPE (indicated by arrowhead in transfected cell). Scale bar = 50 μm. (H) Quantitation of CPE fluorescence intensity in the cell body of transfected neurons. Error bars indicate ± SEM. n (neurons) = 12, CTL siRNA; n = 19, CPE siRNA. ****P < 0.0001 as determined by Student’s t-test.

Article Snippet: Antibodies and Reagents Mouse CPE (BD 610 758) and mouse MAP2 (BD 556 320) primary antibodies were purchased from BD Biosciences (San Jose, CA).

Techniques: In Vivo, Cell Culture, In Vitro, In Utero, Construct, Quantitation Assay, shRNA, Negative Control, Transfection, Fluorescence

Journal: Cerebral Cortex (New York, NY)

Article Title: Cortical Neuron Migration and Dendrite Morphology are Regulated by Carboxypeptidase E

doi: 10.1093/cercor/bhy155

Figure Lengend Snippet: Overexpression of the carboxyl terminus of CPE disrupts neuronal migration and dendritic arborization. (A) Mice at E14.5 were electroporated in utero with constructs as indicated, and brains were analyzed at E17.5. Representative images of coronal brain sections are shown for each condition. White, transfected cells; gray, Hoechst labeling of nuclei. Scale bar = 50 μm. (B) Quantitation of the percentage of transfected cells in each cortical area. Error bars indicate ± SEM. n = 8, GFP; n = 6, CPE-C10 (carboxyl terminal 10 amino acids of CPE). **P < 0.01 as determined by 2-way ANOVA followed by Sidak’s multiple comparisons test. (C) Representative images of transfected cells in the IZ are shown for each condition. Arrows point to unipolar or bipolar cells; arrowheads point to multipolar cells. Scale bar = 20 μm. (D) Quantitation of percentage of unipolar and bipolar cells under each condition. Morphology of transfected cells across the entire IZ was analyzed. Error bars indicate ± SEM. n = 10, GFP; n = 6, CPE-C10. **P < 0.01 as determined by Student’s t-test. (E) Hippocampal neurons were cotransfected with pCAG-mOrange and pEGFP (GFP) or pEGFP-CPE-C10 (CPE-C10) as indicated at DIV 7. Neurons were fixed and immunostained for GFP and MAP2 at DIV 10. Neurons positive for both GFP and mOrange were assessed for Sholl analysis. Representative mOrange fluorescent images of neurons are shown as inverted black images. Scale bar = 50 μm. (F) Sholl analysis of neurons expressing GFP or CPE-C10 on dendrite branching. Gray line indicates P value is at least less than 0.01 as determined by 2-way ANOVA followed by Sidak’s multiple comparisons test. (G) Sholl analysis within 60 μm from soma. Error bars indicate ± SEM. n (neurons) = 95, GFP; n = 90, CPE-C10. *P < 0.05, **P< 0.01, ***P < 0.001 as determined by 2-way ANOVA followed by Sidak’s multiple comparisons test.

Article Snippet: Antibodies and Reagents Mouse CPE (BD 610 758) and mouse MAP2 (BD 556 320) primary antibodies were purchased from BD Biosciences (San Jose, CA).

Techniques: Over Expression, Migration, In Utero, Construct, Transfection, Labeling, Quantitation Assay, Expressing