Journal: bioRxiv

Article Title: Mitochondria dynamics in postmitotic cells drives neurogenesis through Sirtuin-dependent chromatin remodeling

doi: 10.1101/2020.02.07.938985

Figure Lengend Snippet: (A) Representative images of mitochondrial morphology (mito-GFP) in Pax6+ (Apical progenitor in ventricular zone (VZ)) and βIII-tub+ (Newborn neuron in cortical plate (CP)) cell in E16.5 mouse cortex, following in utero retroviral infection at E12.5. Quantified mitochondrial length from two biological replicate experiments. Each data point represents an individual cell average mitochondrial size. ****P < 0.0001; unpaired t test. (B) Schematic of the labeling strategy using photoconverted (PC) histone H2B-mEos4b. Representative images of PC cell labelled with mito-Nep2. (C) Representative images and timeline of PC experiment to determine kinetics of mitochondria dynamics following mitosis in mouse embryonic cortical cells. Quantified mitochondrial length from three biological replicate experiments. Each data point represents an individual cell average mitochondrial size, together with fate marker expression. Red: βIII-tub+ neuron; Green: Tbr2+ intermediate progenitor; Gray: double negative (DN) RGC. (D) Timeline and representative images of PC experiment using M1 and Mdivi-1. (E,G) Quantified mitochondrial length from three biological replicate experiments. (E) 3 hours post-label, (G) 6 hours post-PC. Each data point represents an individual cell average mitochondrial size. **P < 0.01, ***P < 0.001, ****P ≤ 0.0001; Dunnett’s multiple comparisons test. (F,H) Quantification of each cell fate marker+ cells among PC cells from at least four biological replicate experiments. Data are shown as mean ± SEM. **P < 0.01, ***P < 0.001; Dunn’s multiple comparisons test.

Article Snippet: DNA fragment of Cre was prepared by PCR and transferred to retroviral plasmid backbone (a gift from Fred Gage (Addgene plasmid # 49054; http://n2t.net/addgene:49054 ; RRID:Addgene_49054)) by restriction digestion and ligation to obtain a pRetro-CAG-Cre-WPRE.

Techniques: In Utero, Retroviral, Infection, Labeling, Marker, Expressing

Journal: bioRxiv

Article Title: Mitochondria dynamics in postmitotic cells drives neurogenesis through Sirtuin-dependent chromatin remodeling

doi: 10.1101/2020.02.07.938985

Figure Lengend Snippet: (A) Representative images of mitochondrial morphology in SOX2+ (RGC) and βIII-tub+ (Newborn neuron) human ESC-derived cortical cell 3 days post mito-GFP retroviral infection. Quantification of mitochondrial length from two biological replicate experiments. Each data point represents an individual cell average mitochondrial size. ****P < 0.0001; Mann Whitney test. (B) Timeline and representative images of Drp1- and Mff-overexpressing human cortical cells (6 days post Cre-expressing retrovirus infection). Arrow head: Neuron (Tbr1+), Arrow: Progenitor (Sox2+). Quantification of TBR1+ cells among GFP-labeled cells from three biological replicate experiments. Data are shown as mean ± SEM. ****P < 0.001; Dunnett’s multiple comparisons test. (C,D) Representative time-lapse images of mitochondrial dynamics in (C) Symmetric non-neurogenic division and (D) Symmetric neurogenic division. Asterisks indicate tracked cells. (E) Timeline of PC experiment and quantified mitochondrial length from three biological replicate experiments. Each data point represents an individual cell average mitochondrial size. (F,H) Timing of M1 treatment in (F) mouse and (H) human cortical cells. Quantification of βIII-tub+ cells among PC cells from three biological replicate experiments. Data are shown as mean ± SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001; Dunnett’s multiple comparisons test. (G,I) Quantification of mitochondrial length in mouse (G) and human (I) cortical cells from three biological replicate experiments. Each data point represents an individual cell average mitochondrial size. *P < 0.05, **P < 0.01, ****P < 0.0001; Dunn’s multiple comparisons test.

Article Snippet: DNA fragment of Cre was prepared by PCR and transferred to retroviral plasmid backbone (a gift from Fred Gage (Addgene plasmid # 49054; http://n2t.net/addgene:49054 ; RRID:Addgene_49054)) by restriction digestion and ligation to obtain a pRetro-CAG-Cre-WPRE.

Techniques: Derivative Assay, Retroviral, Infection, MANN-WHITNEY, Expressing, Labeling

Journal: Methods in Molecular Biology

Article Title: Stem Cell Assays

doi: 10.1007/978-1-59745-536-7

Figure Lengend Snippet: Fig. 4. (A) Analysis of sulfated glycosaminoglycan (s-GAG) synthesis by human embryoid body (EB)-derived chondrogenic cells cultured in EB outgrowth and high-density micromass (HDMM) along the course of differentiation. Bone morphogenetic protein-2 BMP-2 stimulation of chondrogenic differenti- ation in EB outgrowth culture system displays acute decrease in ratio of s-GAG/DNA from day 7 onward, whereas in HDMM culture system, BMP-2 induces time-dependent increase in ratio of s-GAG/DNA up to day 14, before it decreases, there- after indicating hypertrophic maturation. Values are calculated as means ± SD from duplicate analysis of at least two independent experiments. (B) Alcian blue staining also indicates marked enhancement in s-GAG deposition in the HDMM culture, particularly in the regions of nodular growth.

Article Snippet: Blyscan Sulfated Glycosaminoglycan (s-GAG) Assay kit (Biocolor, Newtownabbey, Ireland).

Techniques: Derivative Assay, Cell Culture, Staining

Journal: Methods in Molecular Biology

Article Title: Stem Cell Assays

doi: 10.1007/978-1-59745-536-7

Figure Lengend Snippet: Fig. 4. Basophilic extracellular matrix (ECM) deposition characteristic of neocar- tilage from ES cell derived EBs in PEGDA hydrogel. EBs were cultured in chondro- genic medium in presence of TGF- 1 for 17 days and stained with Safranin-O and Masson’s trichrome for glycosaminoglycan and collagen detection, respectively.

Article Snippet: Blyscan Sulfated Glycosaminoglycan (s-GAG) Assay kit (Biocolor, Newtownabbey, Ireland).

Techniques: Derivative Assay, Cell Culture, Staining