Journal: Nutrients

Article Title: Asiatic Acid Prevents the Deleterious Effects of Valproic Acid on Cognition and Hippocampal Cell Proliferation and Survival

doi: 10.3390/nu8050303

Figure Lengend Snippet: The number of Ki-67 positive cells in the SGZ of the hippocampal dentate gyrus. Ki-67 positive cells were stained in the SGZ of the dentate gyrus (green; ( A )). All nuclei were counterstained with propidium iodide (red; ( B ) and figure merged ( C )). The number of proliferating cells in animals receiving only VPA was significantly lower (* p < 0.05) than vehicle, Asiatic acid and Asiatic acid plus VPA groups, while Asiatic acid and Asiatic acid plus VPA groups were significantly higher when compared to the vehicle group (# p < 0.05). One-way ANOVA with LSD post hoc test was used to compare between all groups ( D ).

Article Snippet: Sections ware fixed in 0.5% paraformaldehyde (pH 7.4) for 3 min and then sections were incubated with primary antibody monoclonal mouse Ki-67 (1:150, Vector Laboratory, Inc., Burlingame, CA, USA) at room temperature for 1 h. Sections were then incubated with secondary antibody Alexa 488 rabbit anti mouse IgG (1:300, Invitrogen, Carlsbad, CA, USA) for 40 min and counter stained with propidium iodide (PI) (1:6000, Sigma Aldrich, Inc., St. Louis, MO, USA) for 30 s and mounted in glycerol.

Techniques: Staining

Journal: International Journal of Medical Sciences

Article Title: LAP2 Isoform Profile in Heart Ageing and in Cardiac Cell Proliferation and Differentiation: Input From CRISPR-Cas9-mediated LAP2a Knockdown in H9C2

doi: 10.7150/ijms.114095

Figure Lengend Snippet: Characterisation of H9C2 proliferating clones whose genome was modified within the Tmpo gene sequence by CRISPR-Cas9 to reduce or extinguish LAP2a protein expression. ( A ) Schema of the Tmpo gene and LAP2a protein domains. The following are indicated: the number of base pairs targeted by one of the SgRNAs within the Tmpo gene and the amino acid position; the mutations induced by our SgRNA (at protein level) for the four H9C2 clones selected as effectively edited to trigger a premature stop codon; the names of the WT +/+ (unedited CRISPR clones) and of the LAP2a +/- and LAP2a -/- clones considered throughout this study. ( B ) Western blots are shown for whole protein extracts of naive H9C2 and CRISPR clones (WT (21E10, 21B1, 22A11); LAP2a +/- (21H4, 22G2, 22G3) and LAP2a -/- (22B3)), as indicated. Proteins of interest were detected using anti LAP2a Ab (middle panel) and anti TMPO Ab (lower panel). Red Ponceau staining (upper panel) was used to normalize ECL signals. ( C ) The graphs show the ECL signal quantification of western blots (arbitrary units; a.u) after revelation with anti TMPO Ab as shown in 1B. The graphs present the individual values and means ± s.e.m. (N= 2 to 5 independent samples per clone). * p<0.05, ** p<0.01, **** p<0.0001 (Mann Whitney test). ( D ) Immunofluorescence of CRISPR clones (WT (22A11, 21B1), LAP2a -/- (22B3) and LAP2a +/- (21H4)), using a rabbit Ab to detect LAP2a (red), phalloidin to label cytoplasmic actin (green) and DAPI to label nuclear DNA (blue). Scale bar = 50 um. ( E ) The graphs represent the % of cells with either a negative (-) or relatively higher or lower (+, ++, +++) mean signal (a.u) as detected by immunofluorescence when using a rabbit anti LAP2a Ab, as shown in 1D. For the analysed experiment (N = 1), the total n (numbers of cell nuclei) were 184, 321, 174 and 216 for the clones 22B3, 22A11, 21H4 and 21B1, respectively. ( F ) The graph represents the cell doubling time (mean value ± s.e.m.) calculated for naive H9C2 cells, WT CRISPR clones, LAP2a +/- and LAP2a -/- CRISPR edited clones, as indicated. The individual values and means ± s.e.m are given. (N = 4 to 8 independent experiments per clone). * p<0.05; **** p<0.0001. (Mann Whitney test) ( G ) The graph represents the amount of cells that were either positively (green) or negatively (black) stained in situ by immunofluorescence for the proliferation marker Ki67. For the analysed experiment (N = 1), the total n (numbers of cells) were 499, 562 and 647 for the WT, LAP2a +/- and LAP2a -/- clones, respectively. * p<0.05. *** p<0.001 (Chi square test for a contingency table).

Article Snippet: We used the following primary antibodies, according to the manufacturer's instructions: mouse anti-Actin alpha 1 cardiac muscle antibody (Ab) (33-32R, Novus Biological, BioTechne France), mouse anti-Histone H3 Ab (1G1, sc-517576; Santa Cruz Biotechnology, Germany), rabbit anti-Ki67 Ab (NB500-170; Novus Biological, BioTechne France), mouse anti-Lamin A/C Ab (4C11; Cell Signaling Technology, USA); rabbit anti-TMPO/LAP2 Ab (14651-1-AP; Proteintech Europe, United Kingdom) which recognizes all TMPO isoforms (a,b,g); mouse anti-Myosin-2 Ab (MF20, 14-6503-80, Life Technologies Europe, United Kingdom), rabbit anti-LAP2 alpha Ab (IQ175; Immuquest, Europe, United Kingdom); rabbit anti-Lamin B1 Ab ; rabbit anti-LEMD2 Ab (HPA017340, Merck Sigma, France), mouse anti-cardiac troponin T monoclonal Ab (13-11; MA512960 , Life Technologies, France) and phalloidin (P2141-Sigma).

Techniques: Clone Assay, Modification, Sequencing, CRISPR, Expressing, Western Blot, Staining, MANN-WHITNEY, Immunofluorescence, In Situ, Marker

Journal:

Article Title: Puma is required for p53-induced depletion of adult stem cells

doi: 10.1038/ncb2100

Figure Lengend Snippet: Depletion of adult neural stem cells in the dentate gyrus of p53TSD/− mice, which is suppressed by Puma-deficiency. (a) Representative images of sections taken from the brains of 30-day-old p53+/+-, p53+/−-, p53TSD/−-and p53TSD/− Puma−/−-littermates that were immunochemically stained for Ki67, doublecortin and NeuN. (b) Sections from a were immunochemically stained for Sox2 and Glial fibrillary acidic protein (GFAP) to identify the neural stem cells and neurons. Scale bar, 50 μm.

Article Snippet: The primary antibodies used were mouse anti-neuronal-specific nuclear protein (NeuN) antibody (1:200; Chemicon), goat anti-DCX (1:250; Santa Cruz Biotechnology), rabbit anti-Ki67 (1:500; Vector Laboratories), chicken anti-GFAP (1:1000; Chemicon) and rabbit anti-Sox2 (1:200; Chemicon).

Techniques: Staining