Journal: Science advances

Article Title: RNF220-mediated K63-linked polyubiquitination stabilizes Olig proteins during oligodendroglial development and myelination.

doi: 10.1126/sciadv.adk3931

Figure Lengend Snippet: Fig. 5. RNF220 regulates K63-linked polyubiquitination of Olig1 and Olig2. (A) Co-IP analyses of the interaction between endogenous RNF220 and Olig1 or Olig2 in forebrains with the indicated antibodies. IgG, immunoglobulin G. (B) Ubiquitination analyses of polyubiquitinated Olig1 and Olig2 in the forebrains of P7 RNF220-WT (n = 3) and RNF220-cKO (n = 3) mice, and bar graphs (mean ± SD) show normalized polyubiquitination levels against respective expression in the WT controls. (C) Analyses of ubiquitination levels of Olig1 and Olig2 in the forebrains of P60 RNF220-iWT (n = 3) and RNF220-icKO (n = 3) mice with tamoxifen treatment or not, and bar graphs (mean ± SD) show normalized polyubiquitination levels against respective expression in the WT controls without tamoxifen treatment. (D) Ubiquitination analyses of RNF220-mediated polyubiquitination of Olig1 and Olig2 in the presence of WT or indicated ubiquitin mutants in HEK293 cells. HA, hemagglutinin. (E) Ubiquitination analyses of RNF220-mediated polyubiquitination of Olig1 and Olig2 with purified proteins as indicated in vitro. (F) Schematic diagram showing all lysine sites in mouse Olig1 and Olig2 proteins and the lysine sites targeted by RNF220 are highlighted in red. (G) Ubiquitination analyses of RNF220-mediated polyubiquitination of WT or Olig1/2 mutants in HEK293 cells as indicated. IP, immunoprecipitation; WCL, whole-cell lysate; TAM, tamoxifen. Statistical analyses are compared to respective control with Mann-Whitney U test with Bonferroni correction in (D) and unpaired Student’s t test in (F). n.s. (not significant), P > 0.05; **P < 0.01.

Article Snippet: The construct containing mouse Olig1 cDNA was purchased from OriGene (MR203390).

Techniques: Co-Immunoprecipitation Assay, Ubiquitin Proteomics, Expressing, Purification, In Vitro, Immunoprecipitation, Control, MANN-WHITNEY

Journal: Science advances

Article Title: RNF220-mediated K63-linked polyubiquitination stabilizes Olig proteins during oligodendroglial development and myelination.

doi: 10.1126/sciadv.adk3931

Figure Lengend Snippet: Fig. 6. RNF220 maintains Olig1 and Olig2 proteins stabilization through regulating their ubiquitination. (A) WB analyses of protein expression of WT or Olig1/2 mutants when coexpressed with WT RNF220 or mutants lacking ubiquitin E3 ligase activity in HEK293 cells. (B) WB analyses of endogenous protein expression of RNF220, Olig1, and Olig2 in OPC and OL cells, isolated as in fig. S1A, from the brains of P7 RNF220-WT (n = 3), RNF220-cHet (n = 3), and RNF220-cKO (n = 3) mice, and bar graphs (mean ± SD) show normalized levels against respective protein expression in the WT controls. (C) WB analyses of endogenous protein expression of RNF220, Olig1, and Olig2 in isolated OPC from the brains of P60 RNF220-iWT (n = 3) and RNF220-icKO (n = 3) mice with tamoxifen treatment or not, and bar graphs (mean ± SD) show normalized levels against respective protein expression from the WT control without tamoxifen treatment. (D) Cycloheximide chase analyses of protein half-lives of endogenous Olig1 and Olig2 in MOPC cells overexpressing WT RNF220 or RNF220W539R mutant lacking E3 ligase activity, and broken line graphs (mean ± SD) show normalized levels against respective protein expression from the control without cycloheximide treatment. ΔR, ΔRING; CHX, cycloheximide. Statistical analyses are compared to respective control with Mann-Whitney U test with Bonferroni correction. n.s. (not significant), P > 0.05; *P < 0.05; **P < 0.01.

Article Snippet: The construct containing mouse Olig1 cDNA was purchased from OriGene (MR203390).

Techniques: Ubiquitin Proteomics, Expressing, Activity Assay, Isolation, Control, Mutagenesis, MANN-WHITNEY

Journal: Science advances

Article Title: RNF220-mediated K63-linked polyubiquitination stabilizes Olig proteins during oligodendroglial development and myelination.

doi: 10.1126/sciadv.adk3931

Figure Lengend Snippet: Fig. 7. Leukodystrophy-related mutations impair RNF220 regulation on Olig protein stabilization, oligodendroglial differentiation, and myelination. (A) Ubiq- uitination analyses of polyubiquitinated Olig1 and Olig2 when coexpressed with WT RNF220 or leukodystrophy-related missense mutants, RNF220R363Q and RNF220R365Q, in HEK293 cells. (B) WB analyses of endogenous Olig1 and Olig2 expression in MOPC cells overexpressing WT RNF220 or leukodystrophy-related missense mutants, RN- F220R363Q and RNF220R365Q. (C) Representative T2-weighted mouse brain MRI scanning of P60 RNF220-RR (n = 3), RNF220-RQ (n = 3), and RNF220-QQ (n = 3) mice. The arrows indicate the corpus callosum. (D) Representative images showing myelin staining (BG) in corpus callosum regions of P60 RNF220-RR (n = 3), RNF220-RQ (n = 3), and RNF220-QQ (n = 3) mice. (E) Electron microscopy images of the corpus callosum transverse sections from P60 RNF220-RR (n = 4), RNF220-RQ (n = 4), and RNF220-QQ (n = 4) mice. Scale bars, 2 μm. Bar graphs (mean ± SD) show quantification of the number and percentage of myelinated axons, and scatterplots show g-ratio relative to axon diameter. (F) Immunofluorescence staining assays of BrdU and Sox10 in the corpus callosum regions of P3 RNF220-RR (n = 3), RNF220-RQ (n = 3), and RNF220-QQ (n = 3) mice. Scale bars, 50 μm. (G) Bar graphs (mean ± SD) show the quantification of BrdU+Sox10+ cells, Ki67+PDGFRα+ cells on P3, and CC1+Sox10+ cells on P21. Statistical analyses are compared to respective control with Mann-Whitney U test with Bonferroni correction or unpaired Student’s t test. n.s. (not significant), P > 0.05; **P < 0.01.

Article Snippet: The construct containing mouse Olig1 cDNA was purchased from OriGene (MR203390).

Techniques: Expressing, Staining, Electron Microscopy, Immunofluorescence, Control, MANN-WHITNEY

Journal: Science advances

Article Title: RNF220-mediated K63-linked polyubiquitination stabilizes Olig proteins during oligodendroglial development and myelination.

doi: 10.1126/sciadv.adk3931

Figure Lengend Snippet: Fig. 8. Leukodystrophy-related RNF220 mutation knock-in mice show impaired regulation of learning and memory behaviors and of Olig proteins. (A to C) Behavioral tests of novel object recognition (A), three-chamber sociability, and social novelty [(B) and (C)], for P60 RNF220-RR (n = 15), RNF220-RQ (n = 15), and RNF220QQ (n = 15) mice. (A) Bar graphs (mean ± SD) show the percentage of spending time close to new object and logarithm of discrimination index. (B) Bar graphs (mean ± SD) show the percentage of spending time close to inanimate ball and animated stranger mouse and logarithm of social ratio. (C) Bar graphs (mean ± SD) show the percentage of spending time close to new and familiar animated strangers and logarithm of social ratio. (D) Ubiquitination analyses of polyubiquitinated Olig1 and Olig2 in the forebrains of P7 RNF220-RR (n = 3) and RNF220-QQ (n = 3) mice, and bar graphs (mean ± SD) show normalized polyubiquitination levels against respective expression in the WT controls. (E) WB analyses of endogenous protein expression of Olig1 and Olig2 in OPC and OL cells, isolated as in fig. S1A, from the brains of P7 RNF220-RR (n = 3), RNF220-RQ (n = 3), and RNF220QQ (n = 3) mice, and bar graphs (mean ± SD) show normalized levels against respective protein expression in the WT controls. Statistical analyses are compared to respective control with Mann-Whitney U test with Bonferroni correction or unpaired Student’s t test. n.s. (not significant), P > 0.05; **P < 0.01.

Article Snippet: The construct containing mouse Olig1 cDNA was purchased from OriGene (MR203390).

Techniques: Mutagenesis, Knock-In, Ubiquitin Proteomics, Expressing, Isolation, Control, MANN-WHITNEY

Journal: Applied Sciences

Article Title: 3D Bioprinting Human Induced Pluripotent Stem Cell-Derived Neural Tissues Using a Novel Lab-on-a-Printer Technology

doi: 10.3390/app8122414

Figure Lengend Snippet: Figure 4. Flow cytometry of 3D bioprinted NPCs after 15 days of culture in vitro (a) βT-III; (b) Olig2; (c) HB9. n = 3 for all groups. One-way ANOVA and Tukey post-hoc analysis was performed for statistical analysis using a confidence level of 95% (p < 0.05) and 99.9% (p < 0.001). * represents p < 0.05 and ** 99.9% p < 0.001. The same data visualized as a scatterplot can be seen in Supplementary Figure S2.

Article Snippet: Flow Cytometry Cell marker expression of the early neuronal markers, βeta tubulin-III (βT-III) ((IC1195C R&D), oligodendrocyte transcription factor 2 (Olig2) (IC2230P R&D systems, Minneapolis, MN, USA), and the protein homeobox 9 (HB9) (bs-11320R, Bioss antibodies), was quantified on day 15.

Techniques: Flow Cytometry, In Vitro

Journal: Cell stem cell

Article Title: OLIG2 Drives Abnormal Neurodevelopmental Phenotypes in Human iPSC-Based Organoid and Chimeric Mouse Models of Down Syndrome

doi: 10.1016/j.stem.2019.04.014

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit anti-OLIG1 , Phosphosolutions , Cat# 1537; RRID: AB_2492192.

Techniques: Virus, shRNA, Recombinant, Gene Expression, Fractionation, Luciferase, Software