Journal: Cells

Article Title: Specificity and Redundancy of Profilin 1 and 2 Function in Brain Development and Neuronal Structure

doi: 10.3390/cells10092310

Figure Lengend Snippet: Mitotic neural precursor cells (NPCs) in the VZ are disarrayed and increased in E11.5 n-dko embryos. ( A ) Immunofluorescence staining of E11.5 cryosections with anti-p-H3 (S10 phospho-histone 3) antibodies, green, to label dividing NPCs, anti-TUB3B (β3-tubulin) antibody, red, to label differentiated neurons on the pial surface, and DAPI, blue, to label cell nuclei, shows a disarrayed layer of dividing NPCs and ectopic p-H3 labeling in the forebrain region of n-dko, as well as n- Pfn1 ko ; Pfn2 het embryos. On the other hand, embryos carrying a single Pfn1 allele, n- Pfn1 het ; Pfn2 ko , appear unaffected. Scale bar: 20 μm. ( B ) Quantification of p-H3 positive cells in the VZ of E11.5 embryos shows increased linear density in n-dko profilin mutants (14.17 ± 0.89/100 μm) compared to controls (8.91 ± 0.20/100 μm, two-sided Welch’s t -test p < 0.001, n = 8/2 sections/embryos for ctrl and n = 11/2 for n-dko). ( C ) Examples of p-H3-labeled condensed and segregated chromatids in NPCs, with the latter indicating the progression of cell division to the telophase stage. ( D ) Quantification of p-H3 + NPCs with segregated chromatids shows a significant 50% decrease in n-dko embryos (9.5% ± 1.87% compared to 19.42% ± 3.4% in controls, two-sided Welch’s t -test, p = 0.0215, n = 8/2 sections/embryos for ctrl and n = 11/2 for n-dko); * p ≤ 0.05, *** p ≤ 0.001. Scale bar: 20 μm.

Article Snippet: Primary antibodies used were anti-phospho-histone 3 (Ser10) rabbit pcl (Upstate/Merck, 06-570, 1:500) and anti-βIII-tubulin mouse mcl (Promega, Madison, WI, USA, G7121, 1:1000).

Techniques: Immunofluorescence, Staining, Labeling

Journal: Cells

Article Title: Specificity and Redundancy of Profilin 1 and 2 Function in Brain Development and Neuronal Structure

doi: 10.3390/cells10092310

Figure Lengend Snippet: Markers of cell division G2 phase and of neural precursor cells are increased in n-dko embryos. ( A ) Western blots of the markers tested on E11.5 embryo head extracts of profilin mutants and controls. Total lane protein quantified by Coomassie staining of the membrane was used for calibration (a sample membrane is shown).Three extracts per genotype were analyzed, as indicated by the color bars. Histograms representing normalized quantification of ( B ) histone 3 Ser10 phosphorylation (p-H3(S10)), a marker of chromosome condensation, ( C ) γ-tubulin (TUBG), ( D ) CDK1 Tyr15 phosphorylation (p-CDK1(Y15)), and ( E ) PPTG1 (also known as securin) show increased expression in neural double profilin knockout (n-dko) embryos and an intermediate increase in the presence of a single Pfn2 allele (n- Pfn1 ko ; Pfn2 het ). The findings point to an arrest of NPCs cell division in the G2-phase. Histograms representing normalized quantification of the intermediate filament proteins ( F ) vimentin (VIM) and ( G ) glial fibrillary acidic protein (GFAP) show higher expression in n-dko embryos and an intermediate increase in n- Pfn1 ko ; Pfn2 het embryos, suggesting an accumulation of cells with NPC identity. For each marker, one-way ANOVA followed by Dunnett’s post hoc test was applied, to compare to ctrl levels; n = 3 per genotype; * p ≤ 0.05, ** p ≤ 0.01.

Article Snippet: Primary antibodies used were anti-phospho-histone 3 (Ser10) rabbit pcl (Upstate/Merck, 06-570, 1:500) and anti-βIII-tubulin mouse mcl (Promega, Madison, WI, USA, G7121, 1:1000).

Techniques: Western Blot, Staining, Marker, Expressing, Knock-Out

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: UBR5-mediated ubiquitination of ATMIN is required for ionizing radiation-induced ATM signaling and function

doi: 10.1073/pnas.1400230111

Figure Lengend Snippet: ATMIN ubiquitination is required for ATM signaling and checkpoint function post IR. (A) G2/M nocodazole trap and quantification of mitotic index in 293T cells transfected with vector (CTR), Flag-ATMIN wild type, or Flag-ATMIN K238R, measured by FACS of phospho-histone 3 (pH3)-positive cells. (B) 53BP1 and pATM immunofluorescence staining of ATMINf/f MEFs transfected with empty vector (ATMINf/f+CTR) or ATMINΔ/Δ MEFs reconstituted with either wild-type (+wtATMIN) or K238R mutant Flag-ATMIN (+ATMIN-K238R). (C) Quantification of 53BP1-positive cells (with at least six distinct foci) from the experiment in B. (D) Western blots of whole-cell lysates from MEFs treated as in B. (E) Radiosensitivity assay of reconstituted MEFs as in B, showing percentage of surviving colonies 7 d after IR. Error bars represent SEM (***P < 0.005, **P < 0.01, *P < 0.05).

Article Snippet: Fixed cells were stained with phospho-Histone 3 antibody and analyzed using a BD Biosciences FACScan.

Techniques: Transfection, Plasmid Preparation, Immunofluorescence, Staining, Mutagenesis, Western Blot