Journal: Acta Neuropathologica Communications

Article Title: Persistent accumulation of unrepaired DNA damage in rat cortical neurons: nuclear organization and ChIP-seq analysis of damaged DNA

doi: 10.1186/s40478-018-0573-6

Figure Lengend Snippet: a-c Representative examples of immunolabeling for γH2AX of cortical neurons in a dissociated neuron preparation ( a ) and cryosections ( b , c ) counterstained with propidium iodide (PI) from irradiated rat ( a , b ) and mouse ( c ) at 15 days post-IR. Note the presence of typical PDDF associated with the nucleolus. d, e Rat cerebral cortex cryosections double immunolabeled for γH2AX and NeuN illustrate the specific localization of PDDF in NeuN-positive neurons at 15 days post-IR. Scale bars: a-c, e, 5 μm; d, 10 μm

Article Snippet: Rabbit polyclonal antibodies anti-histone H2AX phospho-Ser139 (1/200 immunostaining; Novus Biologicals NB100–384), anti-trimethyl-histone H4 (Lys 20) (1/250 immunostaining; Millipore-Upstate 07–463, USA), anti-53BP1 (1/100 immunostaining; Bethyl Laboratories A300-272A, Inc., USA), anti-CTCF (1/100 immunostaining; Millipore-Upstate 07–729, MA, USA), anti- Iba1 (1/500 immunostaining; Wako 019–19,741), anti-GFAP (1/500 immunostaining; Thermo Fisher Scientific PA3–16727), anti-NeuN (1/100 immunostaining; Abcam ab177487) and anti-Histone H3 (1/2000 Western; Thermo Fisher Scientific PA5–16183).

Techniques: Immunolabeling, Irradiation

Journal: Acta Neuropathologica Communications

Article Title: Persistent accumulation of unrepaired DNA damage in rat cortical neurons: nuclear organization and ChIP-seq analysis of damaged DNA

doi: 10.1186/s40478-018-0573-6

Figure Lengend Snippet: a, b Cerebral cortex cryosections double immunolabeled for γH2AX and GFAP ( a ) or Iba1 ( b ) showing the absence of PDDF in an astrocyte and a microglial cell from irradiated rats at 15 days post-IR. Some neuronal nuclei of different size counterstained with DAPI contain γH2AX-positive PDDF. c Cerebral cortex cryosection illustrating a ß-galactosidase-positive senescent microglial cell free of γH2AX-positive PDDF at 15 days post-IR. Scale bars: 10 μm

Article Snippet: Rabbit polyclonal antibodies anti-histone H2AX phospho-Ser139 (1/200 immunostaining; Novus Biologicals NB100–384), anti-trimethyl-histone H4 (Lys 20) (1/250 immunostaining; Millipore-Upstate 07–463, USA), anti-53BP1 (1/100 immunostaining; Bethyl Laboratories A300-272A, Inc., USA), anti-CTCF (1/100 immunostaining; Millipore-Upstate 07–729, MA, USA), anti- Iba1 (1/500 immunostaining; Wako 019–19,741), anti-GFAP (1/500 immunostaining; Thermo Fisher Scientific PA3–16727), anti-NeuN (1/100 immunostaining; Abcam ab177487) and anti-Histone H3 (1/2000 Western; Thermo Fisher Scientific PA5–16183).

Techniques: Immunolabeling, Irradiation

Journal: Acta Neuropathologica Communications

Article Title: Persistent accumulation of unrepaired DNA damage in rat cortical neurons: nuclear organization and ChIP-seq analysis of damaged DNA

doi: 10.1186/s40478-018-0573-6

Figure Lengend Snippet: a, b Representative examples of double immunolabeling for γH2AX and 53BP1 in dissociated cortical neurons from non-irradiated ( a ) and irradiated rats (15d post-IR) ( b ). Some neurons exhibited a PDDF immunolabeled for γH2AX and 53BP1. Scale bar: 5 μm. c Western blot analysis of γH2AX in parietal cortex lysates from non-irradiated and irradiated rats ( n = 3 animals per group). Protein levels of γH2AX were increased upon DNA-damage induced IR. The expression of histone H3 was used as protein loading control, and the fold increase estimated. d Proportion of cortical neurons containing γH2AX-positive PDDF in non-irradiated and irradiated neurons. (*** p < 0.001 by Student’s t test). e Mean number of PDDF per nucleus within the PDDF-containing neuronal population. (* p < 0.05 by Student’s t test). f Distribution of PDDF in three nuclear regions: perinucleolar, nuclear periphery and nuclear interior. Approximately 70% of PDDF were spatially associated with the nucleolus in both non-irradiated and irradiated cortical neurons. g-l Double labeling for γH2AX in combination with 53BP1 ( g , h ), UBF ( j ), B23 ( k ) or histone H4K20me3 ( l ), and for 53BP1 in combination with WRAP53 ( i ) illustrating the concentration of γH2AX, 53BP1 and WRAP53 in PDDF, and the spatial association of PDDF with the nucleolus ( j , k ) and with heterochromatin masses ( l ). g: non-irradiated neuron. h - l : irradiated neurons at 15 days post-IR. Scale bar: 5 μm

Article Snippet: Rabbit polyclonal antibodies anti-histone H2AX phospho-Ser139 (1/200 immunostaining; Novus Biologicals NB100–384), anti-trimethyl-histone H4 (Lys 20) (1/250 immunostaining; Millipore-Upstate 07–463, USA), anti-53BP1 (1/100 immunostaining; Bethyl Laboratories A300-272A, Inc., USA), anti-CTCF (1/100 immunostaining; Millipore-Upstate 07–729, MA, USA), anti- Iba1 (1/500 immunostaining; Wako 019–19,741), anti-GFAP (1/500 immunostaining; Thermo Fisher Scientific PA3–16727), anti-NeuN (1/100 immunostaining; Abcam ab177487) and anti-Histone H3 (1/2000 Western; Thermo Fisher Scientific PA5–16183).

Techniques: Immunolabeling, Irradiation, Western Blot, Expressing, Control, Labeling, Concentration Assay

Journal: Acta Neuropathologica Communications

Article Title: Persistent accumulation of unrepaired DNA damage in rat cortical neurons: nuclear organization and ChIP-seq analysis of damaged DNA

doi: 10.1186/s40478-018-0573-6

Figure Lengend Snippet: a Immunogold electron microscopy for 53BP1 of a typical PDDF (asterisk) in a rat cortical neuron. It is composed of a loosen network of chromatin fibers decorated with gold particles and appears associated with the nucleolus (No) and a heterochromatin mass (Htc). 15 days post-IR. Scale bar: 500 nm Inset: High magnification of 53BP1-immunolabeled chromatin fibers. b In situ electron microscopy transcription assay illustrating the incorporation of 5′-fluorouridine (5’-FU) into nascent RNA after 45 min of the administration of the halogenated nucleotide. Note the absence of 5’-FU incorporation in the PDDF (asterisk) and its incorporation in the transcriptionally active adjacent euchromatin. Rat cortical neuron after 15 days post-IR. Scale bar: 300 nm. c-e Representative example of double immunolabeling for γH2AX and CTCF in an irradiated cortical neuron showing two perinucleolar PDDF. In addition to a diffuse nuclear distribution of CTCF, this insulator protein appears concentrated in numerous microfoci at the periphery of the γH2AX-positive PDDF. 15 days post-IR. Scale bar: 3 μm. f, g Immunogold electron microscopy for the CTCF shows its preferential distribution at the PDDF boundary with euchromatin, although some scattered gold particles appear within the PDDF (asterisk). No: nucleolus. Scale bars: f, 450 nm; g, 250 nm

Article Snippet: Rabbit polyclonal antibodies anti-histone H2AX phospho-Ser139 (1/200 immunostaining; Novus Biologicals NB100–384), anti-trimethyl-histone H4 (Lys 20) (1/250 immunostaining; Millipore-Upstate 07–463, USA), anti-53BP1 (1/100 immunostaining; Bethyl Laboratories A300-272A, Inc., USA), anti-CTCF (1/100 immunostaining; Millipore-Upstate 07–729, MA, USA), anti- Iba1 (1/500 immunostaining; Wako 019–19,741), anti-GFAP (1/500 immunostaining; Thermo Fisher Scientific PA3–16727), anti-NeuN (1/100 immunostaining; Abcam ab177487) and anti-Histone H3 (1/2000 Western; Thermo Fisher Scientific PA5–16183).

Techniques: Electron Microscopy, Immunolabeling, In Situ, Transcription Assay, Irradiation

Journal: Acta Neuropathologica Communications

Article Title: Persistent accumulation of unrepaired DNA damage in rat cortical neurons: nuclear organization and ChIP-seq analysis of damaged DNA

doi: 10.1186/s40478-018-0573-6

Figure Lengend Snippet: Genome wide distribution of γH2AX in rat cortical neurons 15d upon IR shows persistent DNA damage in specific genomic regions. a Table shows the number of total reads, uniquely aligned reads and called peaks for control and irradiated (I1 and I2) rat cortical neurons. Venn diagram shows the overlap (at least one nucleotide) between the called peaks defined in the different conditions. b Read density plots and heatmaps show genome-wide γH2AX distribution in the conditions described in a . c UCSC browser images showing γH2AX binding in different genomic regions close to neuronal specific processes and pathologies. d ChIP-qPCR analysis of CTCF enrichment around several γH2AX binding sites defined by ChIP-seq. Immunoprecipitation with rabbit IgG was performed to ensure antibody specificity. Neg1 and neg2 correspond to genomic regions with undetectable γH2AX binding. Pcdh and krt loci were used as positive CTCF binding sites. Graph represents the results of two independent biological replicates performed in triplicates.

Article Snippet: Rabbit polyclonal antibodies anti-histone H2AX phospho-Ser139 (1/200 immunostaining; Novus Biologicals NB100–384), anti-trimethyl-histone H4 (Lys 20) (1/250 immunostaining; Millipore-Upstate 07–463, USA), anti-53BP1 (1/100 immunostaining; Bethyl Laboratories A300-272A, Inc., USA), anti-CTCF (1/100 immunostaining; Millipore-Upstate 07–729, MA, USA), anti- Iba1 (1/500 immunostaining; Wako 019–19,741), anti-GFAP (1/500 immunostaining; Thermo Fisher Scientific PA3–16727), anti-NeuN (1/100 immunostaining; Abcam ab177487) and anti-Histone H3 (1/2000 Western; Thermo Fisher Scientific PA5–16183).

Techniques: Genome Wide, Control, Irradiation, Binding Assay, ChIP-qPCR, ChIP-sequencing, Immunoprecipitation

Journal: American Journal of Physiology - Renal Physiology

Article Title: Arginase-2 mediates renal ischemia-reperfusion injury

doi: 10.1152/ajprenal.00620.2016

Figure Lengend Snippet: ARG2 expression in mouse kidneys. A–E: immunohistochemical staining for ARG2 on representative kidney sections from mouse kidneys. See results for details.

Article Snippet: Mouse kidney sections were stained with rabbit anti-ARG2 (1:800 and 1:200, respectively, sc-20151, Santa Cruz Biotechnology).

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: American Journal of Physiology - Renal Physiology

Article Title: Arginase-2 mediates renal ischemia-reperfusion injury

doi: 10.1152/ajprenal.00620.2016

Figure Lengend Snippet: ARG2 expression and activity increased after renal IRI. Both left and right renal pedicles were clamped for 28 min in 7- to 9-wk-old anesthetized mice. Plasma and kidney tissue were collected at the indicated time point following ischemia. Kidney function was assessed for BUN (A) and plasma creatinine levels (B). Quantitative RT-PCR was performed on kidney RNA for Arg2 (C), and Arg1 (D) expression. ARG1 (E) and ARG2 (E and F) protein levels were determined using Western blot. E: representative Western blot of samples for the time course in F. Kidney (G) and plasma (H) arginase activity were determined as described in materials and methods. Values are means ± SE for n = 5 mice/time point. *P < 0.05, **P < 0.01, and ***P < 0.005 vs. 0 h.

Article Snippet: Mouse kidney sections were stained with rabbit anti-ARG2 (1:800 and 1:200, respectively, sc-20151, Santa Cruz Biotechnology).

Techniques: Expressing, Activity Assay, Quantitative RT-PCR, Western Blot

Journal: American Journal of Physiology - Renal Physiology

Article Title: Arginase-2 mediates renal ischemia-reperfusion injury

doi: 10.1152/ajprenal.00620.2016

Figure Lengend Snippet: Arg2 deficiency or arginase inhibition improve kidney function after IRI. WT and Arg2−/− mice were subjected to bilateral renal ischemia for 28 min, followed by reperfusion for 24 h (A and B). Mice were treated with vehicle or BEC 18 h before ischemia (C and D). Values are means ± SE for n = 6 mice in each group. *P < 0.05 and **P < 0.01 vs. sham. #P < 0.05 and ##P < 0.01 vs. vehicle-treated or WT IRI.

Article Snippet: Mouse kidney sections were stained with rabbit anti-ARG2 (1:800 and 1:200, respectively, sc-20151, Santa Cruz Biotechnology).

Techniques: Inhibition

Journal: American Journal of Physiology - Renal Physiology

Article Title: Arginase-2 mediates renal ischemia-reperfusion injury

doi: 10.1152/ajprenal.00620.2016

Figure Lengend Snippet: Body weight and blood pressure in experiment group

Article Snippet: Mouse kidney sections were stained with rabbit anti-ARG2 (1:800 and 1:200, respectively, sc-20151, Santa Cruz Biotechnology).

Techniques:

Journal: American Journal of Physiology - Renal Physiology

Article Title: Arginase-2 mediates renal ischemia-reperfusion injury

doi: 10.1152/ajprenal.00620.2016

Figure Lengend Snippet: Arg2 deficiency or arginase inhibition increased kidney NO after IRI. Levels of nitrate and nitrite and Western blot for total and p-eNOS were determined in whole kidney tissue 24 h after IRI in WT and Arg2−/− mice (A) or BEC-treated mice (B). p-eNOS-to-eNOS ratios in each experiment were normalized to the average value for sham WT or vehicle controls and arbitrarily set to 1.0. Values are means ± SE for n = 6 mice in each group. *P < 0.05 and **P < 0.01 vs. sham. #P < 0.05 and ##P < 0.01 vs. vehicle-treated or WT IRI.

Article Snippet: Mouse kidney sections were stained with rabbit anti-ARG2 (1:800 and 1:200, respectively, sc-20151, Santa Cruz Biotechnology).

Techniques: Inhibition, Western Blot

Journal: American Journal of Physiology - Renal Physiology

Article Title: Arginase-2 mediates renal ischemia-reperfusion injury

doi: 10.1152/ajprenal.00620.2016

Figure Lengend Snippet: Arg2 deficiency or arginase inhibition improve kidney oxidative stress after IRI. Levels are shown of TBARS (A and C) and GSH (B and D) in mouse kidney following ischemia in WT and Arg2−/− mice (A and B) or BEC-treated IRI mice (C and D). Values are means ± SE for n = 6 mice in each group. *P < 0.05 vs. sham. #P < 0.05 vs. vehicle-treated or WT IRI.

Article Snippet: Mouse kidney sections were stained with rabbit anti-ARG2 (1:800 and 1:200, respectively, sc-20151, Santa Cruz Biotechnology).

Techniques: Inhibition

Journal: American Journal of Physiology - Renal Physiology

Article Title: Arginase-2 mediates renal ischemia-reperfusion injury

doi: 10.1152/ajprenal.00620.2016

Figure Lengend Snippet: Arg2 deficiency or arginase inhibition increased kidney PGC-1α mRNA after IRI. Quantitative RT-PCR was performed on kidney RNA for PGC-1α expression in WT and Arg2−/− mice (A) or BEC-treated mice (B) following ischemia. Values are means ± SE for n = 6 mice in each group. *P < 0.05 vs. sham. #P < 0.05 vs. vehicle-treated or WT IRI.

Article Snippet: Mouse kidney sections were stained with rabbit anti-ARG2 (1:800 and 1:200, respectively, sc-20151, Santa Cruz Biotechnology).

Techniques: Inhibition, Quantitative RT-PCR, Expressing

Journal: American Journal of Physiology - Renal Physiology

Article Title: Arginase-2 mediates renal ischemia-reperfusion injury

doi: 10.1152/ajprenal.00620.2016

Figure Lengend Snippet: Arg2 deficiency or arginase inhibition increased kidney mitochondrial ATP after IRI. Kidney mitochondria were isolated for measurement of ATP content in WT and Arg2−/− mice (A) or BEC-treated mice (B) following ischemia. Values are means ± SE for n = 6 mice in each group. *P < 0.05 and **P < 0.01 vs. sham. #P < 0.05 vs. vehicle-treated or WT IRI.

Article Snippet: Mouse kidney sections were stained with rabbit anti-ARG2 (1:800 and 1:200, respectively, sc-20151, Santa Cruz Biotechnology).

Techniques: Inhibition, Isolation