Journal: Purinergic Signalling

Article Title: Up-regulation of P2X7 receptors mediating proliferation of Schwann cells after sciatic nerve injury

doi: 10.1007/s11302-015-9445-8

Figure Lengend Snippet: Expression of P2X7R ir in longitudinal sections of normal sciatic nerves. a–c show co-localization of P2X7R ir (red) and S100Aβ ir (green). Note an arrowhead showing a fibre-like structure and an arrow showing a trapezoid structure in a and b; c is the merged image of a and b. Note an arrow indicating a trapezoid structure double labelled by both P2X7R and S100β antibodies (yellow). d–f show co-localization of P2X7R ir (red) and Tuj-1 ir (green). Note an arrow showing a trapezoid structure in d and an arrow showing an axon in e. f is the merged image of d and f; note an arrow indicating a green axon passing through the middle of five trapezoid structures. g–i show co-localization of P2X7R ir (red) and p75NTR ir (green). Note an arrow showing a fibre-like structure in g and h. i is the merged image of g and h. An arrow indicates a double-labelled non-myelinating Schwann cell with P2X7R and p75NTR antibodies (yellow). j–l show co-localization of P2X7R ir (red) and MBP ir (green). Note an arrow showing a fibre-like structure with P2X7R ir, which was not labelled by MBP in l. m–o show co-localization of P2X7R ir (red) and CASPR ir (green). Note that no colocalization of P2X7R ir and CASPR ir was observed. Scale bars in a–c and g–i = 100 μm; scale bars in d–f = 50 μm

Article Snippet: The sections were washed 3–5 min in PBS, and then preincubated in a blocking solution (10 % normal bovine serum, 0.2 % Triton X-100, 0.4 % sodium azide in 0.01 mol/L PBS, pH 7.2) for 30 min followed by incubation with the primary antibodies: P2X7R (1:1000), Alomone, rabbit polyclonal, APR-004; S100β (1:400), Tuj-1 (1:200), p75NTR, PCNA (1:200), MBP (1:200), mouse monoclonal antibodies from Boster, CASPR (1:400) mouse monoclonal antibodies from Santa Cruz, at room temperature overnight.

Techniques: Expressing

Journal: Molecular medicine reports

Article Title: β-Nerve growth factor attenuates hepatocyte injury induced by D-galactosamine in vitro via TrkA NGFR.

doi: 10.3892/mmr.2013.1590

Figure Lengend Snippet: Figure 1. (A) NGF, (B) TrkANGFR and (C) p75NTR protein expression in L-02 cells (immunocytochemistry; magnification, x200). NGF, nerve growth factor; TrkANGFR, tyrosine kinase-A nerve growth factor receptor; p75NTR, p75 pan-neurotrophin receptor.

Article Snippet: Recombinant human β-NGF was obtained from R&D Systems (Minneapolis, MN, USA) and rabbit polyclonal anti-NGF, anti-TrkANGFR and anti-p75NTR antibodies were derived from Boster Bioengineering Limited Company (Wuhan, China).

Techniques: Expressing, Immunocytochemistry

Journal: Molecular medicine reports

Article Title: β-Nerve growth factor attenuates hepatocyte injury induced by D-galactosamine in vitro via TrkA NGFR.

doi: 10.3892/mmr.2013.1590

Figure Lengend Snippet: Figure 3. β-NGF promotes the proliferation of L-02 cells. (A) The effect of β-NGF on the proliferation of L-02 cells injured by 40 mmol/l of D-GalN after 24 h (*P<0.05 vs. D-GalN). (B) The effect of β-NGF promoting the proliferation of L-02 cells was eradicated by an anti-TrkANGFR blocking antibody (*P<0.01 vs. β-NGF + GalN). Con, control; β-NGF, β-nerve growth factor; D-GalN, D-galactosamine; anti-TrkANGFR, anti-tyrosine kinase A nerve growth factor receptor; p75NTR, p75 pan-neurotrophin receptor; sily, silymarin. Values are presented as the mean ± SD (n=4).

Article Snippet: Recombinant human β-NGF was obtained from R&D Systems (Minneapolis, MN, USA) and rabbit polyclonal anti-NGF, anti-TrkANGFR and anti-p75NTR antibodies were derived from Boster Bioengineering Limited Company (Wuhan, China).

Techniques: Blocking Assay, Control

Journal: bioRxiv

Article Title: Macrophage epigenetic memories of early life injury drive neonatal nociceptive priming

doi: 10.1101/2023.02.13.528015

Figure Lengend Snippet: A. Example sorting of peritoneal macrophages from P7 naïve, P35 naïve, and P7 incised animals isolated at P35. B. Principle component analysis from ATAC-seq datasets demonstrating distinct clustering of groups. C. Principle component analysis form the RNA-seq dataset shows similar distinct clustering of groups. D. The top 100 differentially accessible chromatin regions in macrophages in P35 naïve cells compared to P35 primed cells (age matched). E. The top 100 differentially expressed genes (DEGs) for the same conditions. F. A table of factors that had differentially accessible regions and DEGs. NGFR (p75NTR) is highlighted. G. ATAC-Seq indicating higher reads of NGFR (*p=0.001 vs. controls, one-way ANOVA, Tukey’s, n=3-4/group), and ( H ) transcripts per million (TPM) data for RNA-seq (*p=0.008 vs. controls). Data shown as mean +/- SEM.

Article Snippet: Briefly, 10 nM control (siCON), or siRNAs against p75NTR (sip75; Origene SR321107) were incubated with macrophages at day five in macrophage differentiation medium.

Techniques: Isolation, RNA Sequencing Assay

Journal: bioRxiv

Article Title: Macrophage epigenetic memories of early life injury drive neonatal nociceptive priming

doi: 10.1101/2023.02.13.528015

Figure Lengend Snippet: A. Quality control analyses reveal a pure population of cells that are likely macrophages based on open chromatin regions and previous sequencing datasets. B. Animals that received dual incision regardless of treatment with AP or vehicle had a significant reduction of contralateral mechanical withdrawal thresholds (F=15.337) that lasted for at least seven days (**p<0.05 vs. BL, both groups, Two-way RM ANOVA, Tukey’s, n=10-12. C. Contralateral muscle squeezing is affected by the knockout of p75NTR in LysM+ cells following dual incision (F=13.047) on days one and three (*p<0.001 vs. p75fl/fl BL, ^p<0.05 vs. LysM;p75fl/fl, Two-way RM ANOVA, Tukey’s, n=7/group). Data shown as mean +/- SEM.

Article Snippet: Briefly, 10 nM control (siCON), or siRNAs against p75NTR (sip75; Origene SR321107) were incubated with macrophages at day five in macrophage differentiation medium.

Techniques: Control, Sequencing, Knock-Out

Journal: bioRxiv

Article Title: Macrophage epigenetic memories of early life injury drive neonatal nociceptive priming

doi: 10.1101/2023.02.13.528015

Figure Lengend Snippet: A. Schematic of knocking out p75NTR from macrophages following neonatal incision. B. Representative images of p75NTR labeling (green) co-stained with F4/80 (macrophages, white) in the injured hindpaw. Yellow arrows indicate double positive staining. Scale Bar, 25μM. C. There is a significant reduction (F=4.012) in the percentage of double positive cells in LysM;p75fl/fl animals compared to other genotypes (*p<0.05 vs. controls (p75+/fl and p75fl/fl) and LysM;p75+/fl, one way ANOVA, Tukey’s, n=3-5/group). D. We found no effect of tamoxifen prior to the second injury (No Tam vs. BL time points) on paw guarding. Following the second incision there was robust paw guarding (F=26.258), with a faster recovery in the LysM;p75NTRfl/fl mice (**p<0.05 vs. BL, both groups, #p=0.05 vs BL). Two-way RM ANOVA, Tukey’s. E. No effect of tamoxifen on mechanical withdrawal thresholds was found prior to injury (No Tam vs. BL time points), however there was an effect following a second incision (F=40.516). Both groups displayed mechanical sensitivity on days one, three and seven (**p<0.01 vs. BL) but the p75fl/fl genotype continued to display significant sensitivities at day 14 while LysM;p75fl/fl animals did not (p75fl/fl: *p=0.002 vs. BL, LysM;p75fl/fl: p=0.063 vs. BL). At one day following second incision, LysM;p75fl/fl animals were less hypersensitive than controls (^p=0.01 vs. controls). Two-way RM ANOVA, Tukey’s, n=7/group. Data shown as mean +/- SEM.

Article Snippet: Briefly, 10 nM control (siCON), or siRNAs against p75NTR (sip75; Origene SR321107) were incubated with macrophages at day five in macrophage differentiation medium.

Techniques: Labeling, Staining

Journal: bioRxiv

Article Title: Macrophage epigenetic memories of early life injury drive neonatal nociceptive priming

doi: 10.1101/2023.02.13.528015

Figure Lengend Snippet: A. Examples of the protein arrays that were run on the lysates of BMDMs that received a knockdown of p75NTR and were stimulated with indicated factors. B. Heatmap indicating the protein expression detected across each stimulus, n=4/group. C. Examples of significantly regulated proteins detected from the protein array (*p<0.05 vs. siCON+Veh, ^p<0.05 vs. sip75+LPS, #p=0.05 vs. sip75+Veh, ##p>0.05 vs sip75+Veh; one-way ANOVA, Tukey’s or ANOVA on Ranks, Dunn’s. n=4/group). D-F. Representative images of iPSC-derived macrophages, sensory neurons and Rhod-2 responses to ATP. Arrows indicate increased responses. Arrowheads indicated new responses. G. Example calcium transients from iPSC derived sensory neurons in response to ATP or capsaicin stimulation. The arrow indicates when the ATP or capsaicin was added. Scale Bar, 20μM. H. Exposure of iPSC derived sensory neurons to media from macrophages with loss of p75NTR treated with NGF caused decreased responsiveness of neurons to ATP (F=4.257) (*p<0.04 vs. all other conditions, 1-way ANOVA with Tukey’s post hoc, n=15 cells per group). I . No changes in calcium responses to capsaicin (F=2.702) were observed (1-way ANOVA). Data shown as mean +/- SEM or percent change from controls with variance.

Article Snippet: Briefly, 10 nM control (siCON), or siRNAs against p75NTR (sip75; Origene SR321107) were incubated with macrophages at day five in macrophage differentiation medium.

Techniques: Knockdown, Expressing, Protein Array, Derivative Assay

Journal: bioRxiv

Article Title: Macrophage epigenetic memories of early life injury drive neonatal nociceptive priming

doi: 10.1101/2023.02.13.528015

Figure Lengend Snippet: A. Example pictures of the transfection efficiency to knockdown p75NTR in BMDMs. B. Example blots macrophages that were treated with different stimulants and the knockdown of p75NTR. C. A heatmap consisting of all manipulations for the 40 proteins included in the arrays. D. An analysis indicating no difference in the number of cells obtained from the bone marrow of early life incised or naïve or sham animals isolated at P35 (p=0.617 controls vs. n.inc, ANOVA on Ranks, n=16-18/group). “LPS” indicates LPS+IFNγ stimulation, “si” indicates treatment with the siRNA against p75NTR, “Combo” indicates both NGF and BDNF stimulation applied. Data shown as mean +/- SEM.

Article Snippet: Briefly, 10 nM control (siCON), or siRNAs against p75NTR (sip75; Origene SR321107) were incubated with macrophages at day five in macrophage differentiation medium.

Techniques: Transfection, Knockdown, Isolation

Journal: bioRxiv

Article Title: Macrophage epigenetic memories of early life injury drive neonatal nociceptive priming

doi: 10.1101/2023.02.13.528015

Figure Lengend Snippet: A. A schematic indicating timeline for removal and stimulation of bone marrow cells from previously sham operated or incised neonates. B. Analyses of the transcripts from BMDMs after stimulation with LPS+IFNγ (*p<0.05 vs. n.sham no stimulation controls, ^p<0.05 vs. n.sham with stimulation, n=3-8/group, Two-way ANOVA, Tukey’s). C. Schematic of the bone marrow transplant experiments. D. Representative cell sorting of living cells, 45.1+ host remaining cells and 45.2+ transferred cells. E. p75NTR transcripts are increased in animals that received an early life incision versus those that did not (total peritoneal cells). F. After recovery from the BMT, animals display no guarding behaviors prior to incision. Following an incision both n.sham and n.inc transferred groups display an effect of day (F=25.051) on paw guarding (*p<0.001 vs. BL, Two-way RM ANOVA, Tukey’s). G. There was also an effect of day on withdrawal thresholds (F=9.099) that was significant at days one and three in the primed group (*p<0.01 vs. BL) but was delayed in controls with significant reductions at days three and seven (*p<0.01 vs. BL, Two-way RM ANOVA, Tukey’s, n=10-11/group). H. There is no difference between groups for male-driven mechanical withdrawal thresholds but there is an effect of day (F=6.867) that was significant in controls (*p<0.05 vs. BL, Two-way RM ANOVA, Tukey’s, n=6/group). I. In female mechanical withdrawal thresholds there was an interaction effect (F=4.398) with n.inc animals showing hypersensitivity at days one and three (*p<0.05 vs. BL) and controls at day seven (*p=0.002 vs. BL). At days one and seven, primed animals withdraw thresholds are significantly different compared to controls (^p<0.05 vs. controls, Two-way RM ANOVA, Tukey’s, n=4-5/group). Data shown as mean +/- SEM.

Article Snippet: Briefly, 10 nM control (siCON), or siRNAs against p75NTR (sip75; Origene SR321107) were incubated with macrophages at day five in macrophage differentiation medium.

Techniques: FACS

Journal: bioRxiv

Article Title: p57Kip2 regulates embryonic haematopoietic stem cell numbers by controlling the size of the sympathoadrenal progenitor pool

doi: 10.1101/2021.09.13.459835

Figure Lengend Snippet: Immunohistochemistry on cryosections from E11 wild-type embryos. ( A ) Immunostaining for p57Kip2 (red) and CD34 (green) with DAPI nuclear stain (blue). P57Kip2 expression is highlighted in endothelial cells (arrowheads), sub-endothelial mesenchyme (asterisk) and SA cells (arrows). ( B ) Immunostaining for p57Kip2 (red) and Ngfr (green) with DAPI nuclear stain (blue). P57Kip2 expression is highlighted in sub-endothelial mesenchyme (asterisk) and SA cells (arrows). Dashed line shows outline of the aorta. ( C ) Immunostaining for Pdgfrβ (red) with DAPI nuclear stain (blue). Exclusion of Pdgfrβ expression from SA cells is highlighted (arrows). Scale bars indicate 50μm. ( D ) Sorting strategy for AGM subpopulations; HSPCs, CD34+CD45+; endothelial cells (EC), CD34+CD45-; SA cells, Ngfr+Pdgfrβ-; mesenchymal cells (ME), Ngfr-Pdgfrβ+. Gating was based on fluorescence minus one controls (FMOs) as shown in Figure S1. ( E, F ) p57Kip2 mRNA expression (relative to β actin ) by qPCR in subpopulations sorted from E11 and E12 AGMs. n ≥ 3. Histogram represents mean ±SEM.

Article Snippet: The following antibodies were used: CD45.1 (A20), CD45.2 (104), CD34 (RAM34), Pdgfrβ (APB5), CD45 (104), Ngfr (polyclonal; ANT-007-AO; Alomone Labs Ltd).

Techniques: Immunohistochemistry, Immunostaining, Staining, Expressing, Fluorescence

Journal: bioRxiv

Article Title: p57Kip2 regulates embryonic haematopoietic stem cell numbers by controlling the size of the sympathoadrenal progenitor pool

doi: 10.1101/2021.09.13.459835

Figure Lengend Snippet: ( A ) Immunohistochemical staining of E11 wild-type embryo cryosection for Ngfr (green), p57Kip2 (red) and Th (white). Bar represents ventral vs dorsal subdissection. ( B ) t-SNE plots coloured for clusters identified by k-means clustering (left) and for ventral and dorsal origin (right). Violin plots for expression levels of Ngfr ( C ) and haematopoiesis-associated genes Ptprc / CD45 ( D ), Csf1r ( E ), Cx3cr1 ( F ), Runx1 ( G ), Procr / EPCR ( H ), Kit ( I ) and Cd34 ( J ). ( K ) t-SNE coloured for predicted cell cycle phases (top) and the identified clusters before (middle) and after (bottom) cell cycle correction. ( L ) t-SNE plot of remaining 4 clusters following cell cycle correction (bottom) and coloured according to ventral and dorsal origin (top). t-SNE gene expression plots coloured for expression levels of SNS genes Sox10 ( M ), Th ( N ), Phox2a ( O ), Gata3 ( P ), Phox2b ( Q ) and Cdkn1c/p57Kip2 ( R ).

Article Snippet: The following antibodies were used: CD45.1 (A20), CD45.2 (104), CD34 (RAM34), Pdgfrβ (APB5), CD45 (104), Ngfr (polyclonal; ANT-007-AO; Alomone Labs Ltd).

Techniques: Immunohistochemical staining, Staining, Expressing, Gene Expression