Journal: The Journal of biological chemistry

Article Title: Pirh2-dependent DNA damage in neurons induced by the G-quadruplex ligand pyridostatin.

doi: 10.1016/j.jbc.2023.105157

Figure Lengend Snippet: Figure 8. Pirh2 downregulates DDX21 in cultured primary neurons. A, primary cortical neurons were transfected with the DDX21-NeonGreen construct. Cells were fixed and stained with antibodies against nucleolin and MAP2c and the DAPI dye. Note that DDX21-NeonGreen forms puncta. Scale bar rep- resents 5 μm. B, primary cortical neurons were transfected with DDX21-NeonGreen and treated with a vehicle or 2 μM pyridostatin (PDS) overnight. Cells were fixed, stained with antibodies against nucleolin and MAP2c, and imaged with a confocal microscope. The left panel is merged images (MAP2c, nucleolin, DDX21-NeonGreen, and DAPI). Scale bar represents 5 μm. The right three panels are zoomed in from the left panel to demonstrate that, in vehicle- treated neurons, nucleolin forms ring-like structures (the granular component of the nucleolus) and DDX21-NeonGreen forms puncta mostly within the nucleolin rings (the dense fibrillar component of the nucleolus). Note that PDS profoundly alters nucleolin- and DDX21-NeonGreen-positive foci. Scale bar represents 1 μm. C, colocalization of nucleolin and DDX21-NeonGreen was measured from (B). p = 0.014 (t test), 50 vehicle- and 50 PDS-treated neurons were analyzed from three experiments. D, primary cortical neurons were transfected with Neon-Green and BFP or with NeonGreen and BFP-Pirh2, fixed, and stained with antibodies against DDX21 and the SYTOX dye. Note that DDX21-positive puncta are absent in BFP-Pirh2-expressing cell (depicted with arrow). Scale bar represents 5 μm. E, fluorescence intensities of DDX21 were measured in BFP- and BFP-Pirh2-expressing neurons from (D). p = 0.001 (t test), 33 BFP-transfected and 44 BFP-Pirh2-transfected neurons were analyzed from three experiments. BFP, blue fluorescent protein; DAPI, 40,6-diamidino-2- phenylindole.

Article Snippet: Antibodies against DDX21 were from Proteintech (catalog no.: 10528-1-AP).

Techniques: Cell Culture, Transfection, Construct, Staining, Microscopy, Expressing

Journal: The Journal of biological chemistry

Article Title: Pirh2-dependent DNA damage in neurons induced by the G-quadruplex ligand pyridostatin.

doi: 10.1016/j.jbc.2023.105157

Figure Lengend Snippet: Figure 10. Model of Pirh2-dependent DNA damage in neurons induced by the G-quadruplex ligand pyridostatin (PDS). PDS alters transcription in cultured primary neurons, leading to changes in various neuron-specific and neuron-nonspecific pathways. The E3 ubiquitin ligase Pirh2 is upre- gulated and localizes to the nucleolus, resulting in DNA damage as evi- denced by γH2AX upregulation, downregulation of the helicase DDX21, and elevated G4-DNA levels.

Article Snippet: Antibodies against DDX21 were from Proteintech (catalog no.: 10528-1-AP).

Techniques: Cell Culture, Ubiquitin Proteomics

Journal: Communications biology

Article Title: A transition phase in late mouse oogenesis impacts DNA methylation of the early embryo.

doi: 10.1038/s42003-022-04008-1

Figure Lengend Snippet: Fig. 1 Mouse GVOs undergo an NSN-SN transition through an intermediate stage in late oogenesis. a Representative images of the three types of chromatin configurations in mouse germinal vesicle oocytes (GVOs) stained with Hoechst 33342. Scale bar = 20 μm. b Representative images of EU staining of the different types of GVOs. INT-GVOs were further categorized into early, mid, and late depending on the formation of the perinucleolar ring. EU signal intensity was equally adjusted (EU adj.) to compare the EU signals of INT-GVOs. Scale bar = 10 μm. c Quantification of EU signal intensity in NSN-, INT-, and SN-GVOs. EU signals were normalized to NSN-GVOs. Each dot represents a single oocyte (n = 10 for NSN, n = 12 for INT, and n = 15 for SN; data are derived from three independent experiments). The box plot is showing the interquartile (box), median (horizontal line), minimum and maximum values (error bars). Statistical significance was calculated using one-way analysis of variance (ANOVA with Tukey’s multiple comparison test). d Representative immunofluorescence images of the three types of GVOs analyzed for DDX21 (n > 10 for each type from two independent experiments). Scale bar = 10 μm. e Hoechst 33342 staining of NSN-GVOs 0 h and 24 h after isolation, cultured in IVM media supplemented with IBMX. The asterisk indicates the position of the nucleolus. Scale bar = 20 μm. f Quantification of the transition of NSN-GVOs at different timepoints. NSN-GVOs were isolated, sorted, and cultured under maturation inhibiting conditions for 48 h (n = 42 NSN-GVOs from two independent experiments).

Article Snippet: Oocytes were immunostained with antibodies against DDX21 (rabbit polyclonal (1:500), Novus Biologicals), anti-5mC (mouse monoclonal (1:100), Eurogentec), anti-5hmC (rabbit polyclonal (1:100), Active Motif), anti-5fC (rabbit polyclonal (1:500), gift from Yi Zhang), anti-5caC (rabbit polyclonal (1:500), Diagenode).

Techniques: Staining, Derivative Assay, Comparison, Isolation, Cell Culture

Journal: Communications Biology

Article Title: A transition phase in late mouse oogenesis impacts DNA methylation of the early embryo

doi: 10.1038/s42003-022-04008-1

Figure Lengend Snippet: a Representative images of the three types of chromatin configurations in mouse germinal vesicle oocytes (GVOs) stained with Hoechst 33342. Scale bar = 20 μm. b Representative images of EU staining of the different types of GVOs. INT-GVOs were further categorized into early, mid, and late depending on the formation of the perinucleolar ring. EU signal intensity was equally adjusted (EU adj.) to compare the EU signals of INT-GVOs. Scale bar = 10 μm. c Quantification of EU signal intensity in NSN-, INT-, and SN-GVOs. EU signals were normalized to NSN-GVOs. Each dot represents a single oocyte ( n = 10 for NSN, n = 12 for INT, and n = 15 for SN; data are derived from three independent experiments). The box plot is showing the interquartile (box), median (horizontal line), minimum and maximum values (error bars). Statistical significance was calculated using one-way analysis of variance (ANOVA with Tukey’s multiple comparison test). d Representative immunofluorescence images of the three types of GVOs analyzed for DDX21 ( n > 10 for each type from two independent experiments). Scale bar = 10 μm. e Hoechst 33342 staining of NSN-GVOs 0 h and 24 h after isolation, cultured in IVM media supplemented with IBMX. The asterisk indicates the position of the nucleolus. Scale bar = 20 μm. f Quantification of the transition of NSN-GVOs at different timepoints. NSN-GVOs were isolated, sorted, and cultured under maturation inhibiting conditions for 48 h ( n = 42 NSN-GVOs from two independent experiments).

Article Snippet: Oocytes were then fixed with 4% PFA for 20 min at 4 °C, permeabilized with 0.2% TritonX-100 in PBS for 10 min at RT, and then blocked with 0.1% TritonX-100, 1% BSA in PBS overnight at 4 o C. Oocytes were immunostained with antibodies against DDX21 (rabbit polyclonal (1:500), Novus Biologicals), anti-5mC (mouse monoclonal (1:100), Eurogentec), anti-5hmC (rabbit polyclonal (1:100), Active Motif), anti-5fC (rabbit polyclonal (1:500), gift from Yi Zhang), anti-5caC (rabbit polyclonal (1:500), Diagenode).

Techniques: Staining, Derivative Assay, Comparison, Immunofluorescence, Isolation, Cell Culture