Journal: The Journal of Biological Chemistry

Article Title: Epstein-Barr virus suppresses N 6 -methyladenosine modification of TLR9 to promote immune evasion

doi: 10.1016/j.jbc.2024.107226

Figure Lengend Snippet: EBV infection suppresses TLR9 m 6 A modification and expression. A , schematic diagram of the MeRIP-seq protocol used to identify RNA m 6 A profile changes in BJAB cells upon EBV infection. B , GO and KEGG pathway enrichment results for downregulated m 6 A-modified genes upon EBV infection ( p ≤ 0.05, date from our previous study: #GSE133936). C , BJAB cells were infected with 50 MOI EBV for 48 h. TLR9, EBNA1, and GAPDH protein expressions were examined. D , m 6 A -RIP-qPCR analysis of TLR9 mRNA expression in BJAB cells upon EBV infection. Control group was used for negative control. E and F , Flag-TLR9 was pretransfected into HEK293 cells for 6 h, then, Flag-NC, Flag-EBNA1, or Flag-EBNA1△NLS plasmids were respectively transfected for 24 h. The mRNA and protein expressions were measured by RT-qPCR and Western blotting ( E ). The m 6 A abundance of TLR9 mRNA was analyzed by m 6 A -RIP-qPCR (F). Three independent experiments were performed, and data are shown as the mean ± SD. ∗∗ p < 0.01, ∗∗∗ p < 0.001, ns, not significant. EBNA1, Epstein-Barr nuclear antigen 1; EBV, Epstein-Barr virus; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; m 6 A, N 6 -methyladenosine; MeRIP, methylated RNA immunoprecipitation; MOI, multiplicity of infection; TLR, toll-like receptor; RIP, RNA immunoprecipitation.

Article Snippet: The antibodies used in this study were TLR9 (13674S, CST), EBNA1 (BM1083, OriGene), GAPDH (60004-1-Ig, Proteintech), METTL3 (15073-1-AP, Proteintech), m 6 A (202003, Synaptic Systems), SALL1 (AWA56088, Abiowell),PRKN(AWA10174, Abiowell), MID1 (AWA40807, Abiowell), Myc-tag(60003-2-Ig, Proteintech), TLR3 (AWA50624, Abiowell), TLR7 (AWA58104, Abiowell), TLR8 (AWA58112, Abiowell), ALKBH5 (ab69325, Abcam), FTO (27226-1-AP, Proteintech), GFP (YM3124, Immunoway), α-Tubulin (66031-1-Ig, Proteintech), YTHDF1 (17479-1-AP, Proteintech), YTHDF2 (24744-1-AP, Proteintech), YTHDF3 (25537-1-AP, Proteintech), YTHDC1 (14392-1-AP, Proteintech), TBK1 (38066, CST), p-TBK1 (5483, CST), MYD88 (4283, CST), p-P65 (S536) (3033S, CST), p65 (8242, CST), IRF3 (11904, CST), Histone H3 (A2348, Abclonal), ubiquitin (20326, CST), Goat anti-Rabbit IgG H&L (HRP) (511203, ZEN-Bioscience), Goat anti-Mouse IgG H&L (HRP) (511103, ZEN-Bioscience).

Techniques: Infection, Modification, Expressing, Control, Negative Control, Transfection, Quantitative RT-PCR, Western Blot, Virus, Methylation, RNA Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: Epstein-Barr virus suppresses N 6 -methyladenosine modification of TLR9 to promote immune evasion

doi: 10.1016/j.jbc.2024.107226

Figure Lengend Snippet: TLR9 downstream signaling pathways and cytokine expression are regulated by METTL3. A , cellular m 6 A modification levels were assayed in BJAB-shNC and BJAB-shMETTL3 cells by Dot blot analysis. B , the expression levels of TLR9 and METTL3 were determined by Western blotting in BJAB-shNC and BJAB-shMETTL3 cells. C , BJAB-shNC and BJAB-shMETTL3 cells were treated with ActD (5 μg/ml) for 0, 3, and 6 h; TLR9 mRNA levels were measured by RT-qPCR. D , the indicated TLR9 signaling proteins were detected by cytoplasmic and nuclear proteins isolation. GAPDH and Histone H3 were used for cytoplasm and nucleus protein loading controls, respectively. E , BJAB cells were treated with STM2457(10 μM) and CpG-ODN2006 (5 μM) upon 50 MOI EBV infection, respectively. EBV genomes copy number was determined by qPCR. F , primary B cells were treated with STM2457(10 μM) and CpG-ODN2006(5 μM) with or without 50MOI EBV infection for appointed time. The secretion of IFN-α and CXCL10 were assayed by ELISA. These data are shown as the mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ns, not significant. EBNA1, Epstein-Barr nuclear antigen 1; EBV, Epstein-Barr virus; IFN, interferon; m 6 A, N 6 -methyladenosine; MOI, multiplicity of infection; ODN, oligodeoxynucleotide; RT-qPCR, reverse transcription-quantitative PCR; TLR9, toll-like receptor 9.

Article Snippet: The antibodies used in this study were TLR9 (13674S, CST), EBNA1 (BM1083, OriGene), GAPDH (60004-1-Ig, Proteintech), METTL3 (15073-1-AP, Proteintech), m 6 A (202003, Synaptic Systems), SALL1 (AWA56088, Abiowell),PRKN(AWA10174, Abiowell), MID1 (AWA40807, Abiowell), Myc-tag(60003-2-Ig, Proteintech), TLR3 (AWA50624, Abiowell), TLR7 (AWA58104, Abiowell), TLR8 (AWA58112, Abiowell), ALKBH5 (ab69325, Abcam), FTO (27226-1-AP, Proteintech), GFP (YM3124, Immunoway), α-Tubulin (66031-1-Ig, Proteintech), YTHDF1 (17479-1-AP, Proteintech), YTHDF2 (24744-1-AP, Proteintech), YTHDF3 (25537-1-AP, Proteintech), YTHDC1 (14392-1-AP, Proteintech), TBK1 (38066, CST), p-TBK1 (5483, CST), MYD88 (4283, CST), p-P65 (S536) (3033S, CST), p65 (8242, CST), IRF3 (11904, CST), Histone H3 (A2348, Abclonal), ubiquitin (20326, CST), Goat anti-Rabbit IgG H&L (HRP) (511203, ZEN-Bioscience), Goat anti-Mouse IgG H&L (HRP) (511103, ZEN-Bioscience).

Techniques: Protein-Protein interactions, Expressing, Modification, Dot Blot, Western Blot, Quantitative RT-PCR, Isolation, Infection, Enzyme-linked Immunosorbent Assay, Virus, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Epstein-Barr virus suppresses N 6 -methyladenosine modification of TLR9 to promote immune evasion

doi: 10.1016/j.jbc.2024.107226

Figure Lengend Snippet: EBNA1 induces METTL3 protein degradation via the ubiquitination pathway. A , EBNA1 protein levels were detected in BJAB, Raji, and B95.8 cells. B and C , Flag-NC and Flag-EBNA1 were transfected into HEK293 cells. The mRNA and protein levels of METTL3 and EBNA1 were assayed by RT-qPCR ( B ) and Western blotting ( C ). D , The 100 ng, 200 ng, and 500 ng mRNA from HEK293 cells transfected with Flag-EBNA1 (or Flag-NC as negative control) were extracted to detect the total cellular m 6 A modification level by Dot blot. A concentration of 0.02% methylene blue staining was used as a loading control. E and F , HEK293 cells were transfected with EBNA1 plasmids for 36 h, followed by CHX treatment (25 μg/ml) and MG132 (25 μM) for indicated times. Then cellular proteins were collected, and Western blotting was performed. G , GFP-NC and GFP-EBNA1, Flag-METTL3 and plasmids containing various polyubiquitin chains were cotransfected for 48 h. HEK293 cells were treated with MG132 (25 μM) for 4 h before harvest. Lysates were immunoprecipitated with anti-Flag beads. H , HONE-1 cells were transfected with indicated siRNAs for 48 h. The protein expression levels were detected by Western blotting. I and J , HONE-1 cells were transfected with indicated plasmids, then, the mRNA and protein expression of PRKN was measured by RT-qPCR ( I ) and Western blotting ( J ). K , HEK293 cells were transfected with Flag-PRKN and Myc-METTL3 plasmids for 48 h. The cell lysates were immunoprecipitated with anti-Flag beads (or IgG as control). The first lane was an input control without immunoprecipitation. L , Myc-METTL3, Flag-PRNK, and Ub (either WT or K48R) plasmids were cotransfected into HEK293 cells for 48 h. Cell lysates were immunoprecipitated with anti-Myc antibodies. The data are shown as the mean ± SD. ∗∗ p < 0.01, ∗∗∗ p < 0.001, ns, not significant. CHX, cycloheximide; EBNA1, Epstein-Barr nuclear antigen 1; m 6 A, N 6 -methyladenosine; RT-qPCR, reverse transcription-quantitative PCR.

Article Snippet: The antibodies used in this study were TLR9 (13674S, CST), EBNA1 (BM1083, OriGene), GAPDH (60004-1-Ig, Proteintech), METTL3 (15073-1-AP, Proteintech), m 6 A (202003, Synaptic Systems), SALL1 (AWA56088, Abiowell),PRKN(AWA10174, Abiowell), MID1 (AWA40807, Abiowell), Myc-tag(60003-2-Ig, Proteintech), TLR3 (AWA50624, Abiowell), TLR7 (AWA58104, Abiowell), TLR8 (AWA58112, Abiowell), ALKBH5 (ab69325, Abcam), FTO (27226-1-AP, Proteintech), GFP (YM3124, Immunoway), α-Tubulin (66031-1-Ig, Proteintech), YTHDF1 (17479-1-AP, Proteintech), YTHDF2 (24744-1-AP, Proteintech), YTHDF3 (25537-1-AP, Proteintech), YTHDC1 (14392-1-AP, Proteintech), TBK1 (38066, CST), p-TBK1 (5483, CST), MYD88 (4283, CST), p-P65 (S536) (3033S, CST), p65 (8242, CST), IRF3 (11904, CST), Histone H3 (A2348, Abclonal), ubiquitin (20326, CST), Goat anti-Rabbit IgG H&L (HRP) (511203, ZEN-Bioscience), Goat anti-Mouse IgG H&L (HRP) (511103, ZEN-Bioscience).

Techniques: Ubiquitin Proteomics, Transfection, Quantitative RT-PCR, Western Blot, Negative Control, Modification, Dot Blot, Concentration Assay, Staining, Control, Immunoprecipitation, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Epstein-Barr virus suppresses N 6 -methyladenosine modification of TLR9 to promote immune evasion

doi: 10.1016/j.jbc.2024.107226

Figure Lengend Snippet: A working model depicting the main molecular mechanisms about the current study. EBV EBNA1 increases METTL3 protein degradation via K48-linked ubiquitin-proteasome pathway, which mediated by the E3 ligase PRKN, thus inhibits cellular m 6 A modification. Downregulation of METTL3 inhibits TLR9 m 6 A modification. YTHDF1 is an m 6 A “reader” for cellular TLR9 mRNA with m 6 A modification, and YTHDF1 binding increases TLR9 mRNA translation efficiency. EBV infection decreases the m 6 A levels of TLR9 mRNA, thus reduces TLR9 protein levels, as well as TLR9-induced cytokine secretions. EBV, Epstein-Barr virus; m 6 A, N 6 -methyladenosine; TLR9, toll-like receptor 9.

Article Snippet: The antibodies used in this study were TLR9 (13674S, CST), EBNA1 (BM1083, OriGene), GAPDH (60004-1-Ig, Proteintech), METTL3 (15073-1-AP, Proteintech), m 6 A (202003, Synaptic Systems), SALL1 (AWA56088, Abiowell),PRKN(AWA10174, Abiowell), MID1 (AWA40807, Abiowell), Myc-tag(60003-2-Ig, Proteintech), TLR3 (AWA50624, Abiowell), TLR7 (AWA58104, Abiowell), TLR8 (AWA58112, Abiowell), ALKBH5 (ab69325, Abcam), FTO (27226-1-AP, Proteintech), GFP (YM3124, Immunoway), α-Tubulin (66031-1-Ig, Proteintech), YTHDF1 (17479-1-AP, Proteintech), YTHDF2 (24744-1-AP, Proteintech), YTHDF3 (25537-1-AP, Proteintech), YTHDC1 (14392-1-AP, Proteintech), TBK1 (38066, CST), p-TBK1 (5483, CST), MYD88 (4283, CST), p-P65 (S536) (3033S, CST), p65 (8242, CST), IRF3 (11904, CST), Histone H3 (A2348, Abclonal), ubiquitin (20326, CST), Goat anti-Rabbit IgG H&L (HRP) (511203, ZEN-Bioscience), Goat anti-Mouse IgG H&L (HRP) (511103, ZEN-Bioscience).

Techniques: Ubiquitin Proteomics, Modification, Binding Assay, Infection, Virus

Journal: Virology Journal

Article Title: Stable replication of the EBNA1/OriP-mediated baculovirus vector and its application to anti-HCV gene therapy

doi: 10.1186/1743-422X-6-156

Figure Lengend Snippet: A Genomic profile of HCV showing both coding and non-coding genes . B HCV core region target sites and sequences used for the design of shRNAs. C Construction and schematic representation of EBNA1/OriP baculovirus transfer vector expressing HCV core shRNA.

Article Snippet: The primary antibodies used were monoclonal antibodies against EBNA1 (Acris Antibodies GmbH) and G3PDH (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

Techniques: Plasmid Preparation, Expressing, shRNA

Journal: Virology Journal

Article Title: Stable replication of the EBNA1/OriP-mediated baculovirus vector and its application to anti-HCV gene therapy

doi: 10.1186/1743-422X-6-156

Figure Lengend Snippet: Inhibition of HCV RNA by EBNA1/OriP and wild-type baculovirus-mediated sh452 . A Real time PCR analysis of HCV RNA expression after transduction of HCV full replicon cells (NNC#2, 4 × 10 4 cells/well) with an MOI 50 and 100 baculovirus-mediated shRNA. HCV RNA values relative to the scrambled shRNA control are shown. B Inhibition of HCV replication by baculovirus-mediated core shRNAs. Ac-shRNAs were used to infect HCV replicons and intracellular HCV core protein levels measured after 3 days by an HCV protein antigen CLEIA assay. Error bars represent standard errors of the mean from three experiments. * p < 0.01.

Article Snippet: The primary antibodies used were monoclonal antibodies against EBNA1 (Acris Antibodies GmbH) and G3PDH (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

Techniques: Inhibition, Real-time Polymerase Chain Reaction, RNA Expression, Transduction, shRNA

Journal: Virology Journal

Article Title: Stable replication of the EBNA1/OriP-mediated baculovirus vector and its application to anti-HCV gene therapy

doi: 10.1186/1743-422X-6-156

Figure Lengend Snippet: Long-term inhibition of HCV RNA by EBNA1/OriP and wild-type baculovirus-mediated sh452 . A Real-time RT-PCR analysis of HCV RNA expression after transduction of HCV full replicon cells (NNC#2, 4 × 10 4 cells/well) with Ac-shRNA452 (MOI = 100 [circle]), Ac-EP-shRNA452 (MOI = 100 [square]). B The cytotoxicity of Ac-EP-shRNA452 (square) and Ac-shRNA452 (circle) represented as the percentage reduction of viable Huh-7 cells. A cytotoxicity assay was performed using a BrdU Cell Proliferation ELISA kit according to the manufacturer's instructions (Roche Diagnostics GmbH). The toxicity results are representative of three independent experiments.

Article Snippet: The primary antibodies used were monoclonal antibodies against EBNA1 (Acris Antibodies GmbH) and G3PDH (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

Techniques: Inhibition, Quantitative RT-PCR, RNA Expression, Transduction, Cytotoxicity Assay, Enzyme-linked Immunosorbent Assay

Journal: Virology Journal

Article Title: Stable replication of the EBNA1/OriP-mediated baculovirus vector and its application to anti-HCV gene therapy

doi: 10.1186/1743-422X-6-156

Figure Lengend Snippet: Detection of EBNA1 protein and siRNA in Ac-EP-shRNA452 infected cells . A Western blot analysis of EBNA1 expression in baculovirus-infected Huh7 cells. Cell lysates were prepared 7 days and 14 days post-infection from cells infected with different viruses. Lane 1: Ac-sh452; lane 2: Ac-EP-sh452; lane 3: Ac-EP-control-shRNA. B Expression of siRNA by a baculovirus vector. To demonstrate the intracellular expression of the shRNA construct in the respective siRNA, Huh-7 cells were infected with Ac-EP-shRNA452. The mixture was run on a 15% polyacrylamide TBE urea gel after 3, 7, and 14 days.

Article Snippet: The primary antibodies used were monoclonal antibodies against EBNA1 (Acris Antibodies GmbH) and G3PDH (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

Techniques: Infection, Western Blot, Expressing, shRNA, Plasmid Preparation, Construct

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: S100B inhibition reduces behavioral and pathologic changes in experimental traumatic brain injury

doi: 10.1038/jcbfm.2015.165

Figure Lengend Snippet: S100B−/− mice had improved retention memory function after traumatic brain injury (TBI). (A) TBI induced sensorimotor impairments at all time points when compared with sham-injured mice in the beam walk test (***P<0.001, **P<0.01, vs. sham). However, S100B−/− mice failed to show any improvement in sensorimotor function. Analysis by repeated measures two-way analysis of variance (ANOVA) followed by Tukey's post-hoc test. (B) S100B−/− mice showed significant improvement (+P<0.05, vs. S100B+/+) in Discrimination Index in the novel object recognition task. Analysis by one-way ANOVA followed by Tukey's post-hoc test. (Mean±s.e.m.; n=11 to 14/group).

Article Snippet: Neutralizing anti-S100B antibody treatment: Male C57Bl/6 mice (20 to 25 g, 3 months old) were randomized into six groups and administered 10 mg/kg anti-S100B (BL356, Bethyl Laboratories, Montgomery, TX, USA) or an isotype matched IgG (Catalog no. P50-1000, Bethyl Laboratories) antibody at 24 hours and 14 days after injury, via the intraperitoneal route, based on preliminary pharmacokinetic data.

Techniques:

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: S100B inhibition reduces behavioral and pathologic changes in experimental traumatic brain injury

doi: 10.1038/jcbfm.2015.165

Figure Lengend Snippet: S100B−/− mice had reduced lesion volume but no reduction in neuronal cell loss after traumatic brain injury (TBI). (A) Histologic assessment using unbiased stereology showed that S100B+/+ mice developed a large lesion following TBI, whereas S100B−/− mice showed a significant reduction in lesion size (*P<0.05, vs. S100B+/+). Analysis by one-tailed unpaired Student's t-test. (B) Representative images of the cresyl violet-stained sections show the reduction in lesion volume observed in S100B−/− samples. Stereological assessment of surviving neurons using unbiased stereological techniques was performed in the cortex (C), thalamus (D), and cornu ammonis 1 (CA1) (E) subregion of the hippocampus at 28 days after injury. TBI resulted in significant neuronal cell loss in the cortex (*P<0.05, vs. sham), thalamus (*P<0.05 or **P<0.01, vs. sham), and CA1 hippocampal subregion (**P<0.01, vs. sham). S100B−/− mice did not show any improvement in neuronal survival in all the assessed brain regions. Analysis by one-way analysis of variance followed by Tukey's post-hoc test. (Mean±s.e.m.; n=4 to 7/group).

Article Snippet: Neutralizing anti-S100B antibody treatment: Male C57Bl/6 mice (20 to 25 g, 3 months old) were randomized into six groups and administered 10 mg/kg anti-S100B (BL356, Bethyl Laboratories, Montgomery, TX, USA) or an isotype matched IgG (Catalog no. P50-1000, Bethyl Laboratories) antibody at 24 hours and 14 days after injury, via the intraperitoneal route, based on preliminary pharmacokinetic data.

Techniques: One-tailed Test, Staining

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: S100B inhibition reduces behavioral and pathologic changes in experimental traumatic brain injury

doi: 10.1038/jcbfm.2015.165

Figure Lengend Snippet: S100B−/− mice had reduced microglial activation in the injured cortex after traumatic brain injury (TBI). Stereological assessment of microglial cell number and activation phenotype was performed in the cortex at 7 and 28 days after TBI. (A) Representative images of the Iba-1-stained sections show morphologic features of ramified and hypertrophic and bushy (activated) microglia. (B) S100B−/− mice showed statistically significant increases in the numbers of ramified microglia on 28 days after injury (**P<0.01, vs. sham; +P<0.05, vs. S100B+/+, #P<0.05, vs. S100B−/− at 7 days) when compared with sham and S100B+/+ mice (at 28 days) and S100B−/− mice at 7 days. TBI resulted in significant microglial activation at 7 days after injury (*P<0.05, vs. sham) followed by sustained increases in numbers of activated microglia at 28 days (***P<0.001, vs. sham) when compared with sham. S100B−/− mice showed significant attenuation in the numbers of activated microglia at 28 days (+P<0.05, vs.S100B+/+) when compared with S100B+/+ mice. Analysis by one-way analysis of variance followed by Tukey's post-hoc test. (Mean±s.e.m.; n=4 to 7/group).

Article Snippet: Neutralizing anti-S100B antibody treatment: Male C57Bl/6 mice (20 to 25 g, 3 months old) were randomized into six groups and administered 10 mg/kg anti-S100B (BL356, Bethyl Laboratories, Montgomery, TX, USA) or an isotype matched IgG (Catalog no. P50-1000, Bethyl Laboratories) antibody at 24 hours and 14 days after injury, via the intraperitoneal route, based on preliminary pharmacokinetic data.

Techniques: Activation Assay, Staining

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: S100B inhibition reduces behavioral and pathologic changes in experimental traumatic brain injury

doi: 10.1038/jcbfm.2015.165

Figure Lengend Snippet: Systemic administration of a neutralizing S100B antibody improved functional outcomes following traumatic brain injury (TBI). (A) TBI induced significant sensorimotor impairments at all time points when compared with sham-injured mice in the beam walk test (***P<0.001, vs. sham). Anti-S100B immunoglobulin G (IgG)-treated mice exhibited significant improvements in sensorimotor performance at 7, 14, 21, and 28 days (+P<0.05, vs. vehicle; ^P<0.05, vs. IgG control) after injury when compared with vehicle-treated and IgG control-treated mice. Analysis by repeated measures two-way analysis of variance (ANOVA) followed by Tukey's post-hoc test. (B) TBI-induced deficits in retention memory (P<0.001, vs. sham) in the novel object recognition task were significantly attenuated by systemic administration of anti-S100B IgG (++P<0.01, vs. vehicle); data expressed as Discrimination Index analysis by one-way ANOVA followed by Tukey's post-hoc test (mean±s.e.m.; n=8 to 14/injured group, n=6 to 7/sham group).

Article Snippet: Neutralizing anti-S100B antibody treatment: Male C57Bl/6 mice (20 to 25 g, 3 months old) were randomized into six groups and administered 10 mg/kg anti-S100B (BL356, Bethyl Laboratories, Montgomery, TX, USA) or an isotype matched IgG (Catalog no. P50-1000, Bethyl Laboratories) antibody at 24 hours and 14 days after injury, via the intraperitoneal route, based on preliminary pharmacokinetic data.

Techniques: Functional Assay

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: S100B inhibition reduces behavioral and pathologic changes in experimental traumatic brain injury

doi: 10.1038/jcbfm.2015.165

Figure Lengend Snippet: Systemic administration of a neutralizing S100B antibody reduced lesion volume, improved neuronal survival, and attenuated microglial activation in the cortex after traumatic brain injury (TBI). (A) Histologic assessment using unbiased stereology showed that vehicle-treated mice developed a large lesion following TBI, whereas anti-S100B immunoglobulin G (IgG) treatment resulted in significant reduction in lesion size (***P<0.001, vs. vehicle). Systemic administration of IgG control significantly reduced the lesion volume (**P<0.01, vs. vehicle, +P<0.05, vs. anti-S100B IgG treatment). Analysis by one-way analysis of variance (ANOVA) followed by Tukey's post-hoc test (mean±s.e.m.; n=5 to 7/group). (B) Representative images of the cresyl violet-stained sections show the reduction in lesion volume observed in anti-S100B IgG-treated samples. (C) Stereological assessment of surviving neurons was performed using unbiased stereological techniques in the cortex at 28 days after injury. TBI resulted in significant neuronal cell loss in the cortex (**P<0.01, vs. sham). Systemic administration of anti-S100B IgG significantly improved neuronal survival in the cortex (+P<0.05, vs. vehicle) when compared with vehicle-treated samples. Analysis by one-way ANOVA followed by Tukey's post-hoc test (mean±s.e.m.; n=5/group). (D) Stereological assessment of microglial cell number and activation phenotype was performed in the cortex at 7 and 28 days after TBI. No statistically significant increases in the numbers of ramified microglia were observed on 7 and 28 days after injury. TBI resulted in significant and sustained increases in activated microglia at 7 days after injury followed by sustained increases at 28 days (***P<0.001, vs. sham). Systemic administration of anti-S100B IgG significantly attenuated microglial activation at 7 days after injury (+++P<0.001, vs. vehicle; (^^^P<0.001, vs. IgG control) when compared with vehicle- and IgG control-treated samples Furthermore, anti-S100B IgG continued to show significant reduction in the numbers of activated microglia at 28 days postinjury (+P<0.05, vs. vehicle) when compared with vehicle-treated samples. Analysis by one-way ANOVA followed by Tukey's post-hoc test (mean±s.e.m.; n=5/group).

Article Snippet: Neutralizing anti-S100B antibody treatment: Male C57Bl/6 mice (20 to 25 g, 3 months old) were randomized into six groups and administered 10 mg/kg anti-S100B (BL356, Bethyl Laboratories, Montgomery, TX, USA) or an isotype matched IgG (Catalog no. P50-1000, Bethyl Laboratories) antibody at 24 hours and 14 days after injury, via the intraperitoneal route, based on preliminary pharmacokinetic data.

Techniques: Activation Assay, Staining

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: S100B inhibition reduces behavioral and pathologic changes in experimental traumatic brain injury

doi: 10.1038/jcbfm.2015.165

Figure Lengend Snippet: S100B–AGER complexes are not detectable in traumatic brain injury (TBI). Representative micrographs of ipsilateral cortical regions from TBI/sham sections 7 days after injury and positive/negative control sections processed for proximity ligation assays using S100B+AGER primary antibodies (brown) and counterstained with hematoxylin to visualize nuclei (blue).

Article Snippet: Neutralizing anti-S100B antibody treatment: Male C57Bl/6 mice (20 to 25 g, 3 months old) were randomized into six groups and administered 10 mg/kg anti-S100B (BL356, Bethyl Laboratories, Montgomery, TX, USA) or an isotype matched IgG (Catalog no. P50-1000, Bethyl Laboratories) antibody at 24 hours and 14 days after injury, via the intraperitoneal route, based on preliminary pharmacokinetic data.

Techniques: Negative Control, Ligation

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: S100B inhibition reduces behavioral and pathologic changes in experimental traumatic brain injury

doi: 10.1038/jcbfm.2015.165

Figure Lengend Snippet: S100B does not influence microglial activation markers in vitro. Lipopolysaccharide (LPS) (A) and interferon-gamma (IFNγ) (B) stimulation in BV2 microglia significantly increased nitric oxide (NO) production at 24 h (***P<0.001, ****P<0.0001 vs. control). Pretreatment of cells with S100B (1 μm) did not significantly attenuate LPS- or IFNγ-stimulated increases in NO production. Analysis by one-way analysis of variance (ANOVA) followed by Tukey's post-hoc test (mean±s.d.; n=6/group). IFNγ stimulation in primary mouse microglia significantly increased NO (C) and reactive oxygen species (ROS) (D) production at 24 h (***P<0.001, ****P<0.0001 vs. control). Pretreatment of cells with S100B (1 μm) did not significantly attenuate IFNγ-stimulated increases in NO or ROS production. Analysis by one-way ANOVA followed by Tukey's post-hoc test (mean± s.d.; n=3/group).

Article Snippet: Neutralizing anti-S100B antibody treatment: Male C57Bl/6 mice (20 to 25 g, 3 months old) were randomized into six groups and administered 10 mg/kg anti-S100B (BL356, Bethyl Laboratories, Montgomery, TX, USA) or an isotype matched IgG (Catalog no. P50-1000, Bethyl Laboratories) antibody at 24 hours and 14 days after injury, via the intraperitoneal route, based on preliminary pharmacokinetic data.

Techniques: Activation Assay, In Vitro