Journal: The Journal of Experimental Medicine

Article Title: Inherited IL-18BP deficiency in human fulminant viral hepatitis

doi: 10.1084/jem.20190669

Figure Lengend Snippet: Homozygous 40-nt deletion in IL18BP . (A) Pedigree of the family affected by FVH due to HAV. The patient is shown in black, whereas healthy individuals are shown in white. Where available, IL18BP mutation (NM_173042.2:c.508-19_528del) status is indicated in red. M, mutant. (B) Familial segregation of the mutation and its homozygous state in the patient were confirmed by Sanger sequencing. (C) Graph showing the predicted CADD scores and global AFs of the mutation found in the patient with FVH (red circle) and missense variants of IL18BP (blue circles) for which homozygotes were reported in GnomAD. The CADD-MSC score (90% confidence interval) for IL18BP is indicated by a dashed line. (D) The upper panel shows the exons (1–5) of the canonical IL18BP transcript; the bottom panel shows a diagram for IL-18BP. The signal peptide is highlighted in blue; the Ig domain is shown in red. Start and stop codons are indicated by an arrow and an asterisk, respectively. The c.508-19_528del is shown as a dashed box on the mRNA. The locations of IL18BP alleles from GnomAD are also shown on the protein diagram.

Article Snippet: Immunoblotting was performed with primary antibodies against the His-Tag (MA1-21315, 0.5 μg/ml; Thermo Fisher Scientific) and IL-18BP (AF119, 0.5 μg/ml; R&D Systems).

Techniques: Mutagenesis, Sequencing

Journal: The Journal of Experimental Medicine

Article Title: Inherited IL-18BP deficiency in human fulminant viral hepatitis

doi: 10.1084/jem.20190669

Figure Lengend Snippet: Impact of the IL18BP :c.508-19_528del on gene expression and function. (A) RT-qPCR showing IL18BP levels normalized against endogenous GAPDH expression in EBV-B cell lines from six healthy controls (black), the WT sibling (III.3, purple), and heterozygous family members: brother (III.2, red), father (II.4, blue), and mother (II.9, green). Relative IL18BP expression was determined by normalization against the mean value for WT cells, set to 1 (indicated by a dashed line). The values shown are the means of two independent experiments performed in duplicate. (B) Agarose gel electrophoresis showing aberrant splicing of the IL18BP mRNA in 3′ RACE on EBV-B cells from the heterozygous sibling (III.2), relative to a control cell line (C1) and the WT sibling (III.3). HPRT1 was used as the housekeeping gene control. (C) The nested PCR products from B were cloned, and colonies were sequenced. Diagram (left) and percentages (right) of WT (gray) and mutant (M1 in blue, M2 in red, and M3 in green) splice variants of the IL18BP transcript are shown. The start codon is located at position 1, and the stop codon is at 585, shown by an asterisk, on the WT transcript. The polyadenylation site is at position 1,252 and indicated by A n . (D) Expression levels of each splice variant (WT in gray, M1 in blue, M2 in red, and M3 in green) were determined and normalized against endogenous GAPDH expression levels by RT-qPCR on EBV-B cells from two healthy controls (C2 and C3) and family members. Graph shows the copy numbers of the mutant splice variants relative to the mean copy number for the WT allele in EBV-B cells from C2, C3, and III.3, which was set to 1 (indicated by a dashed line). The values are the means ± SEM of two independent experiments performed in duplicate. (E and F) Representative immunoblot images showing levels of the WT and mutant IL-18BP alleles, M1–M3 (E), and four missense alleles from GnomAD (F) in concentrated supernatants from transiently transfected COS7 cells. Immunoblotting was performed with the His tag antibody (top), and the membrane was then stripped and probed with the IL18BP antibody (bottom). (G) IL-18BP bioassay: IFN-γ production was measured in NK-92 cells stimulated with recombinant human IL-12 (100 pg/ml), IL-18 (10 ng/ml), and/or concentrated supernatants (100 µg/ml of total protein) of COS7 cells transiently transfected with either empty vector or the constructs expressing indicated IL-18BP variants. Graph is presented on a logarithmic scale with base of 10. The data are the means ± SEM of two independent experiments performed in duplicate using the supernatants shown in E and F and Fig. S1, F and G.

Article Snippet: Immunoblotting was performed with primary antibodies against the His-Tag (MA1-21315, 0.5 μg/ml; Thermo Fisher Scientific) and IL-18BP (AF119, 0.5 μg/ml; R&D Systems).

Techniques: Gene Expression, Quantitative RT-PCR, Expressing, Agarose Gel Electrophoresis, Control, Nested PCR, Clone Assay, Mutagenesis, Variant Assay, Western Blot, Transfection, Membrane, Bioassay, Recombinant, Plasmid Preparation, Construct

Journal: The Journal of Experimental Medicine

Article Title: Inherited IL-18BP deficiency in human fulminant viral hepatitis

doi: 10.1084/jem.20190669

Figure Lengend Snippet: Liver immunohistochemical profile of the patient. Liver tissue sections from a control individual, an unrelated patient with FVH due to HAV, and the deceased IL-18BP–deficient FVH patient reported in this study were subjected to immunohistochemical staining with the following markers: Hep Par-1, CD8, perforin, CD57, CD68, and IL-18. Representative zoom-in views of the original images at 400× magnification (Fig. S4) are shown. Hep Par-1 staining of IL-18BP–deficient patient’s liver tissue section displayed a background staining of macrophages, with lower intensity than hepatocytes. Some IL-18–positive hepatocytes and macrophages are indicated with blue and red arrows, respectively. Scale bar represents 50 µm.

Article Snippet: Immunoblotting was performed with primary antibodies against the His-Tag (MA1-21315, 0.5 μg/ml; Thermo Fisher Scientific) and IL-18BP (AF119, 0.5 μg/ml; R&D Systems).

Techniques: Immunohistochemical staining, Control, Staining

Journal: The Journal of Experimental Medicine

Article Title: Inherited IL-18BP deficiency in human fulminant viral hepatitis

doi: 10.1084/jem.20190669

Figure Lengend Snippet: IL-18/IL-18BP–mediated hepatotoxicity. (A and B) Coculture of mock- or HAV-infected hepatocytes (HepG2 and Huh7.5 cells) with NK-92 cells pretreated with IL-18, IL-18 + IL-18BP, or IL-18BP. HAV infection efficiencies in HepG2 and Huh7.5 cells were ∼40% and ~100%, respectively (Fig. S5 E; Materials and methods). The relative survival of calcein-AM–stained HepG2 or Huh7.5 cells was calculated based on the measurement of fluorescence retention within cells (A) and the amount of secreted albumin (B). Relative fluorescence and albumin levels were determined by normalization against the mean value for hepatocytes cocultured with NK92 cells without pretreatment (not treated [NT]), set to 100. A decrease in the fluorescence or in albumin levels indicates an increase in NK cell–induced hepatotoxicity. The data shown are the means ± SEM of three independent experiments performed in quadruplicate (n.s., not significant; **, P < 0.01; ***, P < 0.001; one-way ANOVA with Bonferroni correction for multiple comparisons). (C) A proposed model for IL-18BP deficiency underlying FVH. During the course of acute HAV infection in an otherwise healthy individual (left), IL-18 is secreted by macrophages in the liver. This cytokine activates lymphocytes, such as NK cells, inducing IFN-γ production and cytotoxicity to eliminate HAV-infected cells. IFN-γ also induces IL-18BP secretion by hepatocytes, macrophages, and other nonparenchymal cells (endothelial cells, fibroblasts, and hepatic stellate cells), to buffer IL-18 activity. However, in the absence of IL-18BP (right), excessive IL-18 activity leads to uncontrolled, massive immune-mediated hepatotoxicity and severe liver injury, as in the IL-18BP–deficient individual with FVH.

Article Snippet: Immunoblotting was performed with primary antibodies against the His-Tag (MA1-21315, 0.5 μg/ml; Thermo Fisher Scientific) and IL-18BP (AF119, 0.5 μg/ml; R&D Systems).

Techniques: Infection, Staining, Fluorescence, Activity Assay

Journal: Emerging Infectious Diseases

Article Title: Livestock Susceptibility to Infection with Middle East Respiratory Syndrome Coronavirus

doi: 10.3201/eid2302.161239

Figure Lengend Snippet: Presence of MERS-CoV receptor DPP4 (IHC) and of mucosubstances (PAS) in upper and lower respiratory tract tissues from sheep, pigs, llamas, and horses. A) In the nose, DPP4 (red cytoplasmic or membrane staining) was present on the lining epithelium of pigs, llamas, and horses but not sheep. PAS staining (magenta) demonstrated more mucous cells in the lining epithelium of sheep and horses and a layer of mucus on the lining epithelium of the horses. B) DPP4 (red cytoplasmic or membrane staining) was present on the lining epithelium of the trachea, bronchus/bronchioles, and alveoli in the pigs, llamas and horses but not in the sheep. Original magnification ×400 for all images. DPP4, dipeptidyl peptidase-4; IHC, immunohistochemistry; MERS-CoV, Middle East respiratory syndrome coronavirus; PAS, periodic acid–Schiff; term., terminal.

Article Snippet: In brief, we performed DPP4 IHC staining by using 5 μg/mL of polyclonal goat IgG anti-human DPP4 antibody (R&D Systems, Abingdon, UK) and peroxidase-labeled rabbit anti-goat IgG (1:200; DAKO; Agilent Technologies Company, Santa Clara, CA, USA) as a secondary antibody.

Techniques: Membrane, Staining, Immunohistochemistry

Journal: Journal of Pediatric Hematology/Oncology

Article Title: Chemotherapy-induced Alteration of SDF-1/CXCR4 Expression in Bone Marrow–derived Mesenchymal Stem Cells From Adolescents and Young Adults With Acute Lymphoblastic Leukemia

doi: 10.1097/mph.0000000000000220

Figure Lengend Snippet: FIGURE 3. Secretion and expression of SDF-1 in BM-MSCs. A, SDF-1 level was measured by enzyme-linked immunosorbent assay. (a) The bone marrow plasma level of SDF-1 in patients with ALL was significantly increased compared with that in healthy donors and ALL-c (*P < 0.05; n = 10). (b), The same difference was found when SDF-1 levels were measured in the supernatants of MSC cultures after incubation for 72 hours. B, Western blotting was performed to quantify total SDF-1 in the lysates of MSCs. SDF-1 expression in the MSCs of ALL was significantly higher than that in the MSCs of healthy donors and ALL-c. C, The difference was also found at the transcriptional level by real-time PCR (*P < 0.05, n = 10). D, Surface-CD26 expression on the MSCs from ALL patients measured by flow cytometry was significantly lower than that in healthy donors and ALL-c (*P < 0.05; n = 10). E, Total CD26 was measured by Western blot in the MSCs. The fold difference was shown among the 3 types of MSCs. F, The same difference was also found at the transcriptional level (*P < 0.05; n = 10). ALL indicates acute lymphoblastic leukemia; BM-MSC, bone marrow–derived mesenchymal stem cell; GADPH, glyceraldehyde- 3-phosphate dehydrogenase; PCR, polymerase chain reaction.

Article Snippet: Membranes were hybridized with the following primary antibodies: rabbit anti-human CXCR4 (1:1000; Abcam), goat antihuman SDF-1(1:500; R&D Systems), goat anti-human CD26 (1:1000; R&D Systems), and mouse antibody to glyceraldehyde-3-phosphate dehydrogenase (1:4000; Santa Cruz).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Incubation, Western Blot, Real-time Polymerase Chain Reaction, Flow Cytometry, Derivative Assay, Polymerase Chain Reaction

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Active cathepsins B, L, and S in murine and human pancreatitis

doi: 10.1152/ajpgi.00073.2012

Figure Lengend Snippet: Primary antibodies

Article Snippet: K11777 (50–100 nM) abolished GB123 binding. table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Antibody Species Conditions Source Mouse Cat-B (AF965) Goat IP: 1.0 μg, overnight, 4°C R&D Systems (Minneapolis, MN) Mouse Cat-L (AF1515) Goat IP: 1.0 μg, overnight, 4°C R&D Systems (Minneapolis, MN) Human Cat-S (AF1183) Goat IP: 1.0 μg, overnight, 4°C R&D Systems (Minneapolis, MN) Cat-B (S-12 sc-6493) Goat IF: 1:300, overnight, 4°C Santa Cruz Biotechnology (Santa Cruz, CA) Cat-L ( d -20 sc-6501) Goat IF: 1:300, overnight, 4°C Santa Cruz Biotechnology (Santa Cruz, CA) Cat-S (M-19 sc-6505) Goat IF: 1:300, overnight, 4°C Santa Cruz Biotechnology (Santa Cruz, CA) F4/80 Rat IF: 1:200, overnight, 4°C BMA Biomedicals (Augst, Switzerland) LAMP1 Rat IF: 1:300, overnight, 4°C ABR Affinity Bioreagents (Golden, CO) NeuN Mouse IF: 1:500, overnight, 4°C Millipore (Billerica, MA) GFAP (AB5541) Chicken IF: 1:250, overnight, 4°C Millipore (Billerica, MA) Ox42 (M1/70) Rat IF: 1:200, overnight, 4°C BD Pharmigen (San Diego, CA) β-Actin Mouse WB: 1:10,000, overnight, 4°C Sigma-Aldrich (St. Louis, MO) c-Fos Rabbit IH: 1:20,000, overnight, 4°C Chemicon (Temecula, CA) Open in a separate window IF, immunofluorescence; IH, immunohistochemistry; WB, Western blotting; IP, immunoprecipitation.

Techniques:

Journal: bioRxiv

Article Title: Extracellular CIRP as a Novel Endogenous TREM-1 Ligand to Fuel Inflammation

doi: 10.1101/674218

Figure Lengend Snippet: (A) SPR between rmCIRP and rmTREM-1. Anti-his Ab was used to capture rmCIRP-his. rmTREM-1 was injected as an analyte in concentrations of 0 to 500 nM. (B) 1.5 × 10 4 RAW264.7 cells were treated with rmCIRP (5 μg/ml) at 4°C for 10 min, fixed in a nonpermeabilized fashion, and stained with rabbit anti-mouse CIRP Ab, goat anti-mouse TREM-1 Ab, goat anti-CD11b Ab, Cy5-conjugated AffiniPure donkey anti-goat IgG, and Cy3-conjugated AffiniPure F(ab’)2 fragment donkey anti-rabbit IgG. Confocal microscopy images were obtained at using a Zeiss LSM880 confocal microscope equipped with a 63× objective. Images were analyzed and quantified by using the ZenBlue software. ( C) After the staining protocol described in (B) , cell associated fluorescence was measured on Biotek Synergy Neo2 at 566 nm upon excitation at 488 nm ( E 1), at 681 nm after excitation at 630 nm ( E 2), and at 681 nm after excitation at 488 nm ( E 3). The transfer of fluorescence was calculated as FRET units. FRET unit = [ E 3 both − E 3 none ] − [( E 3 Cy5 − E 3 none ) × ( E 2 both / E 2 Cy5 )] − [( E 3 Cy3 − E 3 none ) × ( E 1 both / E 1 Cy3 )]. Data are expressed as means ± SE obtained from three independent experiments, n=9/group. Groups compared by unpaired t-test (*p<0.01 vs. CD11b). (D) A total of 1 × 10 6 /ml RAW264.7 cells were stimulated with rmCIRP (1 µg/ml) for various times. Extracted proteins were immunoprecipitated by using anti-DAP12 Ab, followed by Western blotting using pTyr (4G10) and DAP12 Ab. Extracted proteins obtained from rmCIRP (1 µg/ml for various times) stimulated RAW264.7 cells (1 × 10 6 /ml) were subjected to Western blotting using pSyk, Syk, and β–actin Abs. Representative western blots for phosphotyrosine (4G10), DAP12, pSyk, Syk, and β–actin are shown. (E, F) Each blot was quantified by densitometric analysis. Phosphotyrosine (pDAP12) and pSyk expression in each sample was normalized to DAP12 or Syk or β-actin expression and the mean values of 0 min of rmCIRP-treated groups were standardized as one for comparison. Data are expressed as means ± SE (n=6 samples/group). The groups were compared by one-way ANOVA and SNK method (*p<0.05 vs. rmCIRP at 0 min). (G) RAW264.7 cells were transfected with TREM-1 siRNA, control siRNA, underwent mock transfection, or no transfection. Cells were then stimulated with PBS control or 1μg/ml rmCIRP. After 6 h, TNF-α in the supernatant was analyzed by ELISA. Data are expressed as means ± SE (n=3 samples/group). Multiple groups were compared by one-way ANOVA and Tukey method (*p<0.05 vs. respective PBS group; # p<0.01 vs. rmCIRP-treated non-transfected cells). (H) RAW264.7 cells (1×10 4 cells/ml) were plated in 96-well culture plate and stimulated with PBS or rmCIRP (1 µg/ml). Simultaneously cells were treated with various doses of LP-17 or LP-17-Sc1. After 24 h, TNF-α in culture supernatants at protein level were measured by ELISA. Data are expressed as means ± SE obtained from five independent experiments (n=3-10 wells/group). The groups were compared by one-way ANOVA and SNK method (*p<0.05 vs. PBS; #p<0.05 vs. rmCIRP+PBS). FRET, fluorescence resonance energy transfer; TNF, tumor necrosis factor; DAP12, DNAX activation protein of 12kDa; ELISA, enzyme-linked immunosorbent assay; PBS phosphate buffered saline.

Article Snippet: Antibodies used were as follows: rabbit anti-mouse CIRP Ab (Catalog 10209-2-AP; ProteinTech, Rosemont, IL), goat anti-mouse TREM-1 Ab (Catalog AF1187; R&D Systems), goat anti-CD11b Ab (Catalog MBS420973MyBioSource, San Diego, CA), Cy5-conjugated AffiniPure donkey anti-goat IgG (Code 705-175-147; Jackson ImmunoResearch Laboratories, West Grove, PA) and Cy3-conjugated AffiniPure F(ab’)2 fragment donkey anti-rabbit IgG (Code 711-166-152; Jackson ImmunoResearch Laboratories).

Techniques: Injection, Staining, Confocal Microscopy, Microscopy, Software, Fluorescence, Immunoprecipitation, Western Blot, Expressing, Comparison, Transfection, Enzyme-linked Immunosorbent Assay, Förster Resonance Energy Transfer, Activation Assay, Saline

Journal: bioRxiv

Article Title: Extracellular CIRP as a Novel Endogenous TREM-1 Ligand to Fuel Inflammation

doi: 10.1101/674218

Figure Lengend Snippet: (A) Partial amino acid sequence of murine CIRP highlighting an area of similarity between murine PGLYRP1. Three peptides (M1, M2, and M3) are highlighted from within the CIRP sequence. RAW264.7 cells (1×10 4 cells/ml) were plated in 96-well culture plate and treated with 10 µg/ml of peptides M1, M2, or M3 for 30 min. Cells were then stimulated with rmCIRP (1 µg/ml). After 24 h, TNF-α in culture supernatants at protein level were measured by ELISA. Data are expressed as means ± SE obtained from two independent experiments (n=6 wells/group). The groups were compared by one-way ANOVA and SNK method (*p<0.05 vs. unstimulated cells and #p<0.05 vs. rmCIRP-treated cells). (B) SPR between rmTREM-1 and M3. M3 was injected as an analyte in concentrations of 0 to 20 μM. (C) RAW264.7 cells (1×10 4 cells/ml) were plated in 96-well culture plate and treated with M3 or M3-Sc1 at a dose of 10 µg/ml for 30 min. Cells were then stimulated with PBS or rmCIRP (5 µg/ml) at 4°C for 10 min, fixed in a nonpermeabilized fashion, and stained as described in 1B. FRET analysis was performed as described in 1B. Data are expressed as means ± SE obtained from three independent experiments, n=9/group. Multiple groups were compared by one-way ANOVA and Tukey method (*p<0.05 vs CD11b + rmCIRP, #p<0.05 vs TREM-1 + rmCIRP). (D) To activate RAW264.7 cells through TREM-1, 96-well flat bottom plates were pre-coated with 20 μg/ml of an agonist anti-TREM-1 mAb overnight at 37°C. The wells were washed with sterile PBS and 5 × 10 4 cells/well were plated. Prior to plating, cells were premixed with either PBS control, M3 (10 μg/ml) or scramble M3-Sc1 (10 μg/ml) for 30 min. After plating, TNF-α production was measured in the culture supernatants after an additional 24 h of incubation. Data are expressed as means ± SE. The experiment was performed two independent times with n=5 wells per group. Multiple groups were compared by one-way ANOVA and Tukey method (*p<0.05 vs. uncoated; #p<0.05 vs. TREM-1 Ab + PBS). (E) RAW264.7 cells (1×10 4 cells/ml) were plated in 96-well culture plate and treated with various doses of M3, M3-Sc1, or M3-Sc1 for 30 min. Cells were then stimulated with PBS or rmCIRP (1 µg/ml). After 24 h, TNF-α in culture supernatants at protein level were measured by ELISA. Data are expressed as means ± SE obtained from three independent experiments (n=4 wells/group). The groups were compared by one-way ANOVA and SNK method (*p<0.05 vs. PBS-treated cells; #p<0.05 vs. rmCIRP + PBS). (F) Macrophages from healthy human donors (1×10 4 cells/ml) were plated in 96-well culture plate and treated with various doses of M3 for 30 minutes. Cells were then stimulated with PBS or rmCIRP (1 µg/ml). After 24 h, TNF-α in culture supernatants at protein level were measured by ELISA. Data are expressed as means ± SE (n=5 wells/group). The groups were compared by one-way ANOVA and SNK method (*p<0.05 vs. PBS-treated cells; #p<0.05 vs. rmCIRP + PBS). FRET, fluorescence resonance energy transfer; TNF, tumor necrosis factor; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate buffered saline; PGLYRP1, peptidoglycan recognition protein 1.

Article Snippet: Antibodies used were as follows: rabbit anti-mouse CIRP Ab (Catalog 10209-2-AP; ProteinTech, Rosemont, IL), goat anti-mouse TREM-1 Ab (Catalog AF1187; R&D Systems), goat anti-CD11b Ab (Catalog MBS420973MyBioSource, San Diego, CA), Cy5-conjugated AffiniPure donkey anti-goat IgG (Code 705-175-147; Jackson ImmunoResearch Laboratories, West Grove, PA) and Cy3-conjugated AffiniPure F(ab’)2 fragment donkey anti-rabbit IgG (Code 711-166-152; Jackson ImmunoResearch Laboratories).

Techniques: Sequencing, Enzyme-linked Immunosorbent Assay, Injection, Staining, Sterility, Incubation, Fluorescence, Förster Resonance Energy Transfer, Saline

Journal: bioRxiv

Article Title: Extracellular CIRP as a Novel Endogenous TREM-1 Ligand to Fuel Inflammation

doi: 10.1101/674218

Figure Lengend Snippet: Sepsis and I/R causes an increased release of eCIRP. As the endogenous ligand eCIRP recognizes TREM-1 and activates intracellular signaling molecules DAP12 and Syk, leading to increased expression of pro-inflammatory mediators that cause excessive inflammation and remote tissue injury. eCIRP increases TREM-1 expression, possibly via positive feedback induction. A small peptide M3 derived from human eCIRP abrogates eCIRP-TREM-1 interaction, thereby leading to decreased inflammation and attenuated ALI. I/R, ischemia and reperfusion; DAP12, DNAX activation protein of 12kDa; ALI, acute lung injury.

Article Snippet: Antibodies used were as follows: rabbit anti-mouse CIRP Ab (Catalog 10209-2-AP; ProteinTech, Rosemont, IL), goat anti-mouse TREM-1 Ab (Catalog AF1187; R&D Systems), goat anti-CD11b Ab (Catalog MBS420973MyBioSource, San Diego, CA), Cy5-conjugated AffiniPure donkey anti-goat IgG (Code 705-175-147; Jackson ImmunoResearch Laboratories, West Grove, PA) and Cy3-conjugated AffiniPure F(ab’)2 fragment donkey anti-rabbit IgG (Code 711-166-152; Jackson ImmunoResearch Laboratories).

Techniques: Expressing, Derivative Assay, Activation Assay

Journal: The Journal of Neuroscience

Article Title: A Multilevel Screening Strategy Defines a Molecular Fingerprint of Proregenerative Olfactory Ensheathing Cells and Identifies SCARB2, a Protein That Improves Regenerative Sprouting of Injured Sensory Spinal Axons

doi: 10.1523/JNEUROSCI.1002-13.2013

Figure Lengend Snippet: The 102 selected genes for the siRNA screens of the microarray analysis in the olfactory pathway after peripheral deafferentiation and of cultured OECs

Article Snippet: After 24 h, fibroblasts were fixed with PFA for 30 min and labeled for GFP (AB16901; Millipore Bioscience Research Reagents; 1:1000), followed by a Dyelight-488 (Jackson ImmunoResearch Laboratories; 1:800) secondary antibody and labeled for FZD1 (AF1120; R&D Systems; 1:50), SCARB2 (AF1888; R&D Systems; 1:500), or SERPINI1 (ab33077; Abcam; 1:500) followed by a biotinylated (Vector Laboratories; 1:400) secondary antibody and a Cy3-Streptavidine (Jackson ImmunoResearch Laboratories; 1:800) tertiary antibody.

Techniques: Microarray, Cell Culture, Gene Expression, Coagulation, Binding Assay, Membrane, RNA Binding Assay, Activity Assay

Journal: The Journal of Neuroscience

Article Title: A Multilevel Screening Strategy Defines a Molecular Fingerprint of Proregenerative Olfactory Ensheathing Cells and Identifies SCARB2, a Protein That Improves Regenerative Sprouting of Injured Sensory Spinal Axons

doi: 10.1523/JNEUROSCI.1002-13.2013

Figure Lengend Snippet: Validation of LV-mediated overexpression in skin fibroblasts by qPCR and immunocytochemistry. A, Relative gene expression after transduction of fibroblasts with either LV-NGF or 1 of the 18 constructed LV vectors containing the candidate gene sequences. Gene expression is presented relative to the endogenous expression levels in LV-GFP-transduced fibroblasts (dotted line). Differences in relative expression levels between candidate genes can largely be explained by differences in endogenous expression levels. The value for each condition is the average of at least two separate transduction experiments. B–D, Fibroblasts were transduced with LV-SCARB2 (B), LV-SERPINI1 (C), and LV-FZD1 (D). LV-GFP was used as control. Cells were stained for GFP (green) and SCARB2, SERPINI1, and FZD1, respectively (red). Similar exposure times were used for SCARB2, SERPINI1, and FZD1 stainings for cells transduced with LV vectors containing the respective gene and LV-GFP-transduced cells. Scale bar, 100 μm. E, Western blot of endogenous protein levels for SCARB2 in OECs and fibroblasts and SCARB2 protein level in overexpressing fibroblasts. F, Quantification of the protein levels shown in E after background correction and normalization to the expression of β-actin. Data are presented as the mean ± SEM of two technical replicates normalized to the endogenous levels in OECs. SCARB2 levels were 66% and 271% for endogenous and overexpressing fibroblasts, respectively.

Article Snippet: After 24 h, fibroblasts were fixed with PFA for 30 min and labeled for GFP (AB16901; Millipore Bioscience Research Reagents; 1:1000), followed by a Dyelight-488 (Jackson ImmunoResearch Laboratories; 1:800) secondary antibody and labeled for FZD1 (AF1120; R&D Systems; 1:50), SCARB2 (AF1888; R&D Systems; 1:500), or SERPINI1 (ab33077; Abcam; 1:500) followed by a biotinylated (Vector Laboratories; 1:400) secondary antibody and a Cy3-Streptavidine (Jackson ImmunoResearch Laboratories; 1:800) tertiary antibody.

Techniques: Biomarker Discovery, Over Expression, Immunocytochemistry, Gene Expression, Transduction, Construct, Expressing, Control, Staining, Western Blot

Journal: The Journal of Neuroscience

Article Title: A Multilevel Screening Strategy Defines a Molecular Fingerprint of Proregenerative Olfactory Ensheathing Cells and Identifies SCARB2, a Protein That Improves Regenerative Sprouting of Injured Sensory Spinal Axons

doi: 10.1523/JNEUROSCI.1002-13.2013

Figure Lengend Snippet: Overexpression of six candidate genes in a feeding layer of skin fibroblasts increases neurite length of dissociated adult DRG neurons. A, Representative pictures of adult DRG neurons cultured for 40 h on a feeding layer of fibroblasts expressing GFP, NGF, SCARB2, or MSLN, respectively. Fibroblast feeding layer is labeled with phalloidin (green; left images) and adult DRG are labeled with β3-tubulin (red, left; and white, middle and right). The longest neurite of each neuron was traced using cellomics technology (purple; right). B, Average neurite length of adult DRG neurons on nontransduced fibroblasts, fibroblasts overexpressing NGF, or overexpressing one of the 18 candidate genes. The average length of the longest neurite in each condition was normalized to the average length on a LV-GFP transduced feeding layer (dotted line). Adult DRG cultured on nontransduced fibroblasts showed comparable outgrowth to DRG on LV-GFP transduced fibroblasts. Overexpression of NGF (positive control) and six of the candidate genes resulted in a significant increase in neurite length as follows: NGF (160%, p < 0.001), CX3CL1 (128%; *p < 0.05), FZD1 (118%; ***p < 0.001), LEPRE1 (115%, *p < 0.05), S100A9 (152%, **p < 0.01), SCARB2 (123%, *p < 0.05), and SERPINI1 (135%, **p < 0.01). Overexpression of TIMP2 resulted in a significant decrease in length (90%, *p < 0.05). Overexpression of the other 11 candidate genes had no significant effect on neurite length. Data are presented as the normalized mean ± SEM of the average neurite length. The value for each condition is the average of the wells of at least three different outgrowth experiments, each with a minimum of 2 wells per condition and 75 neurons per well. Scale bar, 100 μm.

Article Snippet: After 24 h, fibroblasts were fixed with PFA for 30 min and labeled for GFP (AB16901; Millipore Bioscience Research Reagents; 1:1000), followed by a Dyelight-488 (Jackson ImmunoResearch Laboratories; 1:800) secondary antibody and labeled for FZD1 (AF1120; R&D Systems; 1:50), SCARB2 (AF1888; R&D Systems; 1:500), or SERPINI1 (ab33077; Abcam; 1:500) followed by a biotinylated (Vector Laboratories; 1:400) secondary antibody and a Cy3-Streptavidine (Jackson ImmunoResearch Laboratories; 1:800) tertiary antibody.

Techniques: Over Expression, Cell Culture, Expressing, Labeling, Positive Control