Journal: International journal of stem cells

Article Title: Usp7 Regulates Glial Lineage Cell-Specific Transcription Factors by Modulating Histone H2B Monoubiquitination.

doi: 10.15283/ijsc23202

Figure Lengend Snippet: Fig. 1. Potential ubiquitin-specific protease (USP) candidates regulating H2B monoubiquitination. (A) The effect of CRISPR/Cas9-mediated knockout (KO) of each USP candidate (Usp7, Usp12, Usp21, and Usp37) on the expression level of histone H2B monoubiquitination (H2Bub1) was estimated by Western blotting. (B) The expression of H2Bub1 was graphically represented. *p＜0.05, ***p＜0.001 by paired t-test. (C) Schematic representation of retinoic acid (RA)-induced P19 cell neuronal differentiation schedule. P19 cells formed aggregates for four days with 10 μM of RA. Aggregates were collected and seeded in cell culture plates on day 4 and harvested at the indicated time points. (D) RA-induced neuronal differentiation of P19 cells at different days. Samples were collected at the indicated time points and the expressions of Oct3/4 and βIII-tubulin protein were analyzed by Western blotting. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was analyzed as a loading control. (E) Fluorescence microscopy of P19 cell aggregates after transfection of Cas9 tagged with red fluorescent protein (RFP) along with single guide RNA (sgRNA) targeting Usp7. Scale bar=100 μm.

Article Snippet: Mouse embryonic carcinoma cell line P19 cells were purchased from the American Type Culture Collection.

Techniques: Ubiquitin Proteomics, CRISPR, Knock-Out, Expressing, Western Blot, Cell Culture, Control, Fluorescence, Microscopy, Transfection

Journal: International journal of stem cells

Article Title: Usp7 Regulates Glial Lineage Cell-Specific Transcription Factors by Modulating Histone H2B Monoubiquitination.

doi: 10.15283/ijsc23202

Figure Lengend Snippet: Fig. 2. Usp7 knockout (KO)-mediated negative regulation of H2B ubiquitination during retinoic acid (RA)-induced neuronal differentiation. (A) The histone H2B monoubiquitination (H2Bub1) protein expression levels in the control and Usp7 KO P19 cells during RA-induced neuronal differentiation analyzed by Western blot. Differentiated samples were collected on the indicated days. The protein expression of H2Bub1 and total H2B were analyzed by Western blot. (B) RA-induced neuronal differentiation in P19 cells. Differentiated samples were collected on the indicated days and the protein expression of Usp7 were analyzed by Western blot. (C) The messenger RNA (mRNA) expression of Usp7 in control P19 cells was estimated by real-time polymerase chain reaction during RA-induced differentiation. Error bars were derived from three independent experiments. N=3. ***p＜0.001.

Article Snippet: Mouse embryonic carcinoma cell line P19 cells were purchased from the American Type Culture Collection.

Techniques: Knock-Out, Ubiquitin Proteomics, Expressing, Control, Western Blot, Real-time Polymerase Chain Reaction, Derivative Assay

Journal: International journal of stem cells

Article Title: Usp7 Regulates Glial Lineage Cell-Specific Transcription Factors by Modulating Histone H2B Monoubiquitination.

doi: 10.15283/ijsc23202

Figure Lengend Snippet: Fig. 3. The expression of transcription factors related to mouse neurogenesis upon Usp7 knockout (KO). (A-C) Rela- tive expression levels of oligodendro- cyte-specific transcription factors (A) Olig2, (B) Sox10, and (C) O4 messe- nger RNA (mRNA) were analyzed by real-time polymerase chain reaction (PCR). Samples were collected on day 9 after differentiation from control and Usp7 KO P19 cells. Results were nor- malized with the level of GAPDH. (D-F) Relative expression of astrocyte- specific transcription factor (D) GFAP, (E) ALDH1L1, and (F) S100β were an- alyzed by real-time PCR. Error bars were derived from three independent experiments. N=3. ***p＜0.001.

Article Snippet: Mouse embryonic carcinoma cell line P19 cells were purchased from the American Type Culture Collection.

Techniques: Expressing, Knock-Out, Real-time Polymerase Chain Reaction, Control, Derivative Assay

Journal: International journal of stem cells

Article Title: Usp7 Regulates Glial Lineage Cell-Specific Transcription Factors by Modulating Histone H2B Monoubiquitination.

doi: 10.15283/ijsc23202

Figure Lengend Snippet: Fig. 4. Knockout (KO) of Usp7 alters histone H2B monoubiquitination (H2Bub1) of genes involved in neuronal differentiation. (A-C) Relative expression levels of neuronal lineage cell-specific transcription factors (A) Ascl1, (B) Ngn1, (C) NeuroD1, and (D, E) marker genes (D) Map2 and (E) βIII-tubulin messenger RNA (mRNA) were analyzed with real-time polymerase chain reaction (PCR). Samples were collected on day 9 after differentiation from control and Usp7 KO P19 cells. Results were normalized with the level of GAPDH. (F-H) Samples were collected on day 9 after differentiation from control and Usp7 KO P19 cells and subjected to chromatin immunoprecipitation analysis with antibodies specific for H2Bub1 and H2B. Immunoprecipitated DNA was quantified by real-time PCR with primers specific for the promoter regions of (F) Olig2, (G) GFAP, and (H) Pax6. Error bars were derived from three independent experiments. N=3. RA: retinoic acid. *p＜0.05, **p＜0.01, ***p＜0.001.

Article Snippet: Mouse embryonic carcinoma cell line P19 cells were purchased from the American Type Culture Collection.

Techniques: Knock-Out, Expressing, Marker, Real-time Polymerase Chain Reaction, Control, Chromatin Immunoprecipitation, Immunoprecipitation, Derivative Assay

Journal:

Article Title: Lsh, an epigenetic guardian of repetitive elements

doi: 10.1093/nar/gkh821

Figure Lengend Snippet: Lsh targets to repetitive sequences. (A) Western analysis of Lsh expression in the embryonal carcinoma cell-line P19 and embryonal fibroblasts (MEFs) by using a specific antibody raised against the C-terminal portion of Lsh. (B) ChIPs were performed on P19 cells using the C-terminal antibody against Lsh or an IgG control. The precipitated material was examined by western analysis using the C-terminal anti-Lsh antibody for detection. (C) ChIPs were performed from P19 chromatin extracts using the C-terminal antibody against Lsh or an IgG control. Precipitated DNA or chromatin input was analyzed for the presence of the indicated repeat sequences. DNA was diluted (1:3) for the detection of linear PCR amplification.

Article Snippet: Embryonal carcinoma cell line P19 was purchased from ATCC and cultured according to the manufacturer's protocol.

Techniques: Western Blot, Expressing, Amplification

Journal: Pharmaceutics

Article Title: Screening for Best Neuronal-Glial Differentiation Protocols of Neuralizing Agents Using a Multi-Sized Microfluidic Embryoid Body Array

doi: 10.3390/pharmaceutics14020339

Figure Lengend Snippet: ( A ) Schematic workflow of neuronal and astrocytic differentiation protocols using retinoic acid (RA) or EC23 as inducers. Diameters of embryoid bodies after one-, two-, three- and four-day treatment with concentrations of non-treated (NT), 0.5 µM, 1.0 µM, and 10 µM of ( B ) RA and ( E ) EC23. Bright-field micrographs of non-treated, ( C ) 10 µM RA-treated, and ( F ) 10 µM EC23-treated P19 embryoid bodies in a microtiter plate after four-day treatment. Scale bar, 100 µm. Viability of embryoid bodies after one-, two-, three- and four days post-treatment with increasing concentrations of ( D ) RA and ( G ) EC23. Statistical significance was tested by using one-way ANOVA (* p < 0.05 and *** p < 0.001), n = 3 ± SD. Bars without * do not represent statistical significance.

Article Snippet: The murine embryonal carcinoma cells line P19 (ATCC, Manasass, VA, USA, CRL-1825) was maintained in Minimum Essential Medium Alpha Modification (α-MEM; Sigma-Aldrich, Vienna, Austria) supplemented with 7.5% newborn calf serum (Sigma-Aldrich, Vienna, Austria), 2.5% fetal bovine serum (Sigma-Aldrich, Vienna, Austria), and 1% antibiotic/antimycotic solution (Sigma-Aldrich, Austria) under standard cell culture conditions at 37 °C in a 5% CO 2 humidified atmosphere.

Techniques: