Journal: The Journal of Neuroscience

Article Title: Proliferating NG2-Cell-Dependent Angiogenesis and Scar Formation Alter Axon Growth and Functional Recovery After Spinal Cord Injury in Mice

doi: 10.1523/JNEUROSCI.3953-16.2017

Figure Lengend Snippet: Primary antibodies used, their concentration, host species, company of origin, and RRID information

Article Snippet: See for details on primary antibodies. table ft1 table-wrap mode="anchored" t5 Table 2. caption a7 Primary antibody: specificity Concentration Host species Vendor Catalog no. RRID CD31: blood vessels 1/2000 Rat BD Pharmingen 551262 RRID: AB_398497 CD68: macrophages 1/500 Rat Serotec (now known as Bio-Rad) MCA1957 RRID: AB_322219 GFAP: astrocytes 1/10,000 Rabbit Dako N1506 RRID: AB_10013482 GSTπ: oligodendrocytes 1/1500 Rabbit Biorbyt orb18037 RRID: AB_10778283 Ki67: proliferating cells 1/200 Rat Dako M7248 RRID: AB_2250503 Laminin: ECM molecule 1/5000 Rabbit Sigma-Aldrich L9393 RRID: AB_477163 Mac1: Cd11b, microglia/macrophages 1/200 Rat DSHB M1/70.15.11.50.2 RRID: AB_2234066 NeuN: neurons 1/10,000 Rabbit Abcam ab177487 RRID: AB_2532109 Neurofilament: axons 1/1000 Chicken Aves Labs NF-H RRID: AB_2313552 NG2: pericytes and oligodendrocyte progenitors 1/200–1/1000 Rabbit Millipore AB5320 RRID: AB_11213678 PDGFRβ: pericytes, fibroblasts 1/500 Rabbit Abcam ab32570 RRID: AB_777165 Open in a separate window Primary antibodies used, their concentration, host species, company of origin, and RRID information Immunofluorescence Sections were rinsed in 0.1 m PBS and blocked for nonspecific antigen binding using BP + or BP3 + for 1 h. Next, sections were incubated in primary antibodies overnight at 4°C.

Techniques: Concentration Assay

Journal: The Journal of Neuroscience

Article Title: Proliferating NG2-Cell-Dependent Angiogenesis and Scar Formation Alter Axon Growth and Functional Recovery After Spinal Cord Injury in Mice

doi: 10.1523/JNEUROSCI.3953-16.2017

Figure Lengend Snippet: Changes in spinal cord NG2 expression over time after proliferating NG2+ cell ablation. A, B, Representative images of NG2/Neutral Red immunolabeling at 7 dpi in Tg+Sal and Tg+GCV spinal cords. Insets show regions in red boxes at higher power. C, Quantification of NG2+ glia and NG2+ pericytes at 7 dpi in Tg+Sal versus Tg+GCV C5 spinal cord sections. Branched NG2+ glia were significantly reduced by ∼40% and NG2+ pericytes were significantly reduced by ∼50% in Tg+GCV mice compared with Tg+Sal mice. D, E, Representative images of NG2 immunolabeling at 11 dpi in Tg+Sal and Tg+GCV spinal cords. F, Percentage area of NG2 immunoreactivity in the lesion border of Tg+GCV mice was significantly increased compared with control mice at 11 dpi, then decreased significantly to control levels by 21 dpi. G, H, Representative images of NG2 immunolabeling at 21 dpi in Tg+Sal and Tg+GCV spinal cords. I, Oligodendrocytes immunolabeled for GSTπ in 11 and 21 dpi tissue were quantified in lesion borders. At 11 dpi, Tg+GCV lesion borders had significantly more oligodendrocytes compared with Tg+Sal. Asterisks in images indicate lesions. Scale bars: A, B, 100 μm; D–H, 50 μm. *p < 0.05; **p < 0.01.

Article Snippet: See for details on primary antibodies. table ft1 table-wrap mode="anchored" t5 Table 2. caption a7 Primary antibody: specificity Concentration Host species Vendor Catalog no. RRID CD31: blood vessels 1/2000 Rat BD Pharmingen 551262 RRID: AB_398497 CD68: macrophages 1/500 Rat Serotec (now known as Bio-Rad) MCA1957 RRID: AB_322219 GFAP: astrocytes 1/10,000 Rabbit Dako N1506 RRID: AB_10013482 GSTπ: oligodendrocytes 1/1500 Rabbit Biorbyt orb18037 RRID: AB_10778283 Ki67: proliferating cells 1/200 Rat Dako M7248 RRID: AB_2250503 Laminin: ECM molecule 1/5000 Rabbit Sigma-Aldrich L9393 RRID: AB_477163 Mac1: Cd11b, microglia/macrophages 1/200 Rat DSHB M1/70.15.11.50.2 RRID: AB_2234066 NeuN: neurons 1/10,000 Rabbit Abcam ab177487 RRID: AB_2532109 Neurofilament: axons 1/1000 Chicken Aves Labs NF-H RRID: AB_2313552 NG2: pericytes and oligodendrocyte progenitors 1/200–1/1000 Rabbit Millipore AB5320 RRID: AB_11213678 PDGFRβ: pericytes, fibroblasts 1/500 Rabbit Abcam ab32570 RRID: AB_777165 Open in a separate window Primary antibodies used, their concentration, host species, company of origin, and RRID information Immunofluorescence Sections were rinsed in 0.1 m PBS and blocked for nonspecific antigen binding using BP + or BP3 + for 1 h. Next, sections were incubated in primary antibodies overnight at 4°C.

Techniques: Expressing, Immunolabeling, Control

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: CHAF1A Blocks Neuronal Differentiation and Promotes Neuroblastoma Oncogenesis via Metabolic Reprogramming.

doi: 10.1002/advs.202005047

Figure Lengend Snippet: Figure 4. CHAF1A gene expression and pathway analyses of NB cells and patients. a) Left: overlap of differentially expressed genes (DEGs, |(fc)| > = 1.25, FDR < 0.1) between control (CHAF1A OFF) and CHAF1A-overexpressing SHEP cells (CHAF1A ON, 96 h) and CHAF1A-correlated genes (FDR < 0.1) in patient cohort 1 (n = 249) and 2 (n = 648). Right: GO pathway enrichment analysis of the overlapped genes (ranked by −Log10FDR, FDR<0.05). b) Work flow of the metabolomics analysis: global metabolomics analysis was performed by GC-MS and LC-MS (DiscoveryHD4 platform, Metabolon Inc.) in CHAF1A-overexpressing SHEP cells (DOX 1 μg mL−1 for 0, 24, and 72 h, n = 5). c) Metabolite enrichment analysis depicts the pathways significantly up- and down-regulated by CHAF1A (DOX 24 h, FDR < 0.25); Benjamini–Hochberg corrected two-sided homoscedastic t-test. d) Left: schematic presentation (redrawn from Gamble et al.[52]) of the polyamine pathway with metabolite changes in SHEP cells with or without CHAF1A overexpression for 24 h (red = upregulated metabolites, p ≤0.05; blue = downregulated metabolites, p ≤0.05). Right: polyamine levels in SHEP cells with or without CHAF1A overexpression for 24 h. Data are mean ± SD (n = 5). e) Targeted polyamine analysis in IMR32 cells with conditional KD of CHAF1A (DOX 1 μg mL−1 for 5 days). Differential metabolites (FDR < 0.25) are presented in the heatmap (yellow = upregulated; blue = downregulated) (n = 4). f) Polyamine synthetic and catabolic gene expression in SHEP cells with or without CHAF1A overexpression (24 h). Data are mean ± SD (n = 2); *p < 0.05, **p < 0.01, ***p < 0.001; two-sided unpaired t-test. g) Polyamine gene expression in patients with high and low CHAF1A expression (average CHAF1A mRNA expression ± 1SD, Figure 1) in patient cohorts 1 and 2. Data are mean ± SEM (n = 44 in cohort 1 and n = 107 in cohort 2); *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; two-sided unpaired t-test. h) ODC1 activity in SHEP, GIMEN, and NGP cells with or without CHAF1A overexpression (8 h). One unit is defined as the fluorescence change per minute. Data are normalized by the protein amount and presented as the fold change compared to control (mean ± SD, n = 2); *p < 0.05, **p < 0.01; two-sided unpaired t-test. MTA = 5’-methylthioadenosine; AdoMet = S- (5’-Adenosyl)-L-methionine; AdoHyc = S-(5′-Adenosyl)-L-homocysteine; FC = fold change.

Article Snippet: [26] Primary antibodies: CHAF1A (1:1000, Abcam, 126 625), Cell Cycle Regulation Antibody Sampler Kit (1:500 or 1:1000, Cell Signaling, 9932), RAS (1:1000, cell signaling, 3965), and MYCN (1:500, cell signaling, 9405), ODC1 (1:400, Novus Biologicals, NBP2-32887), HA Tag (1:2000, Abcam, ab9110), CypB (1:1000, 20 361, or 1:500, Santa Cruz Biotechnology, 130 626), β-actin (1:10000, Sigma, A5316), and GAPDH (1:10 000, Proteintech, 10494-AP).

Techniques: Gene Expression, Control, Gas Chromatography-Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Over Expression, Expressing, Activity Assay

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: CHAF1A Blocks Neuronal Differentiation and Promotes Neuroblastoma Oncogenesis via Metabolic Reprogramming.

doi: 10.1002/advs.202005047

Figure Lengend Snippet: Figure 5. Inhibition of polyamine synthesis restores neuronal differentiation. a,b) Neurite length and TUJ1 immunofluorescence staining in NGP- CHAF1A cells treated with RA (5 𝜇м), DOX (1 μg mL−1), and DFMO (0.5 mм) for 72 h. Data are mean ± SEM (n > 300); ****p < 0.0001; one-way ANOVA with Tukey’s multiple comparisons test. Scale bar = 50 μm. c) Cell cycle analysis of NGP-CHAF1A cells treated with DOX (1 μg mL−1) or DFMO (0.5 mм) for 72 h. Data are mean ± SD (n = 2); *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; two-way ANOVA with Tukey’s multiple comparisons test. d) Neuronal outgrowth of IMR32 CHAF1A KD cells in the presence or absence of ODC1 overexpression. Neurite length is quantified using Image J2 and presented as mean ± SEM (n > 300); ****p < 0.0001; one-way ANOVA with Tukey’s multiple comparisons test. Scale bar = 50 μm. e) Cell cycle analysis of IMR32 CHAF1A KD cells in the presence or absence of ODC1 overexpression. Data are mean ± SD (n = 2); ****p < 0.0001; two-way ANOVA with Tukey’s multiple comparisons test. f) Cell viability of LAN5, IMR32, CHLA255, and SK-N-AS cells treated with increasing concentrations of DFMO single agent, RA single agent, and their combination (combo). Cell viability of LAN5 shCTRL and shCHAF1A cells treated with increasing doses of RA. Data are mean ± SD (n = 3). †synergy with CI < 1; **p < 0.01; ***p < 0.001; two-sided unpaired t-test. g) Apoptosis of LAN5, IMR32, CHLA255, and SK-N-AS cells treated with DFMO, RA, and combo (IC50–75). Apoptosis of LAN5 shCTRL and shCHAF1A cells treated with RA (IC50). Data are mean ± SD (n = 3); * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; two-way ANOVA with Tukey’s multiple comparisons test. h) Polyamine metabo- lites in LAN5 cells treated with DFMO (1 mм), RA (10 𝜇м), and combo for 5 days (n = 4). Metabolites with FDR < 0.05 in at least one comparison are shown in the heatmap (red = upregulated; blue = downregulated); two-way ANOVA with original FDR method of Benjamini and Hochberg. The relative abundance of putrescine and spermidine are presented in box and whiskers plots. # indicates FDR < 0.05. i) Top, scheme of RA+DFMO study in LAN5 luc orthotopic xenograft model. Mice were treated with vehicle (1% methylcellulose, p.o., b.i.d., 5 days per week), RA (p.o., 40 mg kg−1 b.i.d.,

Article Snippet: [26] Primary antibodies: CHAF1A (1:1000, Abcam, 126 625), Cell Cycle Regulation Antibody Sampler Kit (1:500 or 1:1000, Cell Signaling, 9932), RAS (1:1000, cell signaling, 3965), and MYCN (1:500, cell signaling, 9405), ODC1 (1:400, Novus Biologicals, NBP2-32887), HA Tag (1:2000, Abcam, ab9110), CypB (1:1000, 20 361, or 1:500, Santa Cruz Biotechnology, 130 626), β-actin (1:10000, Sigma, A5316), and GAPDH (1:10 000, Proteintech, 10494-AP).

Techniques: Inhibition, Staining, Cell Cycle Assay, Over Expression, Comparison

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: CHAF1A Blocks Neuronal Differentiation and Promotes Neuroblastoma Oncogenesis via Metabolic Reprogramming.

doi: 10.1002/advs.202005047

Figure Lengend Snippet: Figure 6. CHAF1A is a direct target of MYCN. a,b) mRNA and protein expression of CHAF1A in TET-21/N cells when MYCN is turned offupon DOX treatment (2 μg mL−1, 24–96 h). GAPDH is used as housekeeping gene, CypB as protein loading control. Data are mean ± SD (n = 2–3); * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; one-way ANOVA with Dunnett’s multiple comparisons test. c) MYCN ChIP-qPCR assays in TET-21/N cells. Input (white bars) and MYCN-ChIP (black bars) samples were analyzed by qPCR using specific primers for CHAF1A (Table S5, Supporting Information). Data from two independent experiments are shown (mean ± SEM, n = 2). d) mRNA expression of CHAF1A, MYCN, MYCN targets and polyamine genes in LAN5 cells upon CHAF1A KD (DOX 1 μg mL−1 for 2–5 days). GAPDH served as control. Mean ± SD (n = 3); * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; two-way ANOVA with Dunnett’s multiple comparisons test. e) Protein expression of CHAF1A, MYCN, and ODC1 in LAN5 cells upon CHAF1A KD (DOX 1 μg mL−1 for 0–10 days). CypB served as protein loading control. Mean ± SD (n = 2); * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; one-way ANOVA with Dunnett’s multiple comparisons test. f) Correlation of CHAF1A, MYCN, and MYCN signature scores in patient cohort 1 (n = 249) and cohort 2 (n = 648). Signatures are defined in the methods section. FC = fold change.

Article Snippet: [26] Primary antibodies: CHAF1A (1:1000, Abcam, 126 625), Cell Cycle Regulation Antibody Sampler Kit (1:500 or 1:1000, Cell Signaling, 9932), RAS (1:1000, cell signaling, 3965), and MYCN (1:500, cell signaling, 9405), ODC1 (1:400, Novus Biologicals, NBP2-32887), HA Tag (1:2000, Abcam, ab9110), CypB (1:1000, 20 361, or 1:500, Santa Cruz Biotechnology, 130 626), β-actin (1:10000, Sigma, A5316), and GAPDH (1:10 000, Proteintech, 10494-AP).

Techniques: Expressing, Control, ChIP-qPCR

Journal: eLife

Article Title: Translational control of polyamine metabolism by CNBP is required for Drosophila locomotor function

doi: 10.7554/eLife.69269

Figure Lengend Snippet: Cellular nucleic acid-binding protein (CNBP) and ornithine decarboxylase (ODC) content correlates with polyamine levels in muscle cells from DM2 patients. ( A ) Immunoblot showing the levels of both human CNBP (hCNBP) and human ODC (hODC) proteins in three DM2 or control muscle cells, with the corresponding band quantification normalized on the loading control (IMAGE J 1.50i; quantification data in source data for western blot [WB] quantification). Vinculin, loading control. A.U., arbitrary unit. (B) Columns represent putrescine content in muscle cells obtained from four DM2 patients ( CTRL ) or from four healthy individuals ( DM2 ), expressed in ng/mg of tissue. Error bars represent SEM; **p>0.001, in unpaired t-test. Full data in . Figure 5—source data 1. Putrescine content quantification as shown in .

Article Snippet: Primary antibodies were: anti-CNBP goat (1:500; Abcam, Ab 48027); anti-Actin goat (1:1000; Santa Cruz, sc-1616); anti-ODC rabbit (1:500; Enzo Life Science, BML-PW8880-0100); anti-CNBP mouse (1:1000; generated by Agrobio for this work), anti-HA HRP (1:500; Santa Cruz, sc-7392), anti-GFP mouse (1:500; Santa Cruz, sc-9996); anti-FLAG HRP (1:1000; Sigma, A8592); anti-Vinculin mouse (1:1000; Santa Cruz, sc-73614); anti-Tubulin mouse (1:7000; Sigma, T-5168).

Techniques: Binding Assay, Western Blot

Journal: eLife

Article Title: Translational control of polyamine metabolism by CNBP is required for Drosophila locomotor function

doi: 10.7554/eLife.69269

Figure Lengend Snippet: Schematic representation of the mechanism of action of CNBP on muscle function in both Drosophila and humans. CNBP binds ornithine decarboxylase (ODC) mRNA and regulates its translation, leading to increased polyamine content. Lack of CNBP impairs locomotor function through ODC-polyamine downregulation.

Article Snippet: Primary antibodies were: anti-CNBP goat (1:500; Abcam, Ab 48027); anti-Actin goat (1:1000; Santa Cruz, sc-1616); anti-ODC rabbit (1:500; Enzo Life Science, BML-PW8880-0100); anti-CNBP mouse (1:1000; generated by Agrobio for this work), anti-HA HRP (1:500; Santa Cruz, sc-7392), anti-GFP mouse (1:500; Santa Cruz, sc-9996); anti-FLAG HRP (1:1000; Sigma, A8592); anti-Vinculin mouse (1:1000; Santa Cruz, sc-73614); anti-Tubulin mouse (1:7000; Sigma, T-5168).

Techniques:

Journal: eLife

Article Title: Translational control of polyamine metabolism by CNBP is required for Drosophila locomotor function

doi: 10.7554/eLife.69269

Figure Lengend Snippet:

Article Snippet: Primary antibodies were: anti-CNBP goat (1:500; Abcam, Ab 48027); anti-Actin goat (1:1000; Santa Cruz, sc-1616); anti-ODC rabbit (1:500; Enzo Life Science, BML-PW8880-0100); anti-CNBP mouse (1:1000; generated by Agrobio for this work), anti-HA HRP (1:500; Santa Cruz, sc-7392), anti-GFP mouse (1:500; Santa Cruz, sc-9996); anti-FLAG HRP (1:1000; Sigma, A8592); anti-Vinculin mouse (1:1000; Santa Cruz, sc-73614); anti-Tubulin mouse (1:7000; Sigma, T-5168).

Techniques: Injection, Mutagenesis, Sequencing