Journal: bioRxiv

Article Title: HMGB1 acts as an agent of host defense at the gut mucosal barrier

doi: 10.1101/2023.05.30.542477

Figure Lengend Snippet: a, Immunostaining for HMGB1 (yellow) and bisbenzimide H 33258 (Hoescht, blue) in Carnoy’s fixed proximal colon sections from HMGB1 WT and HMGB1 ΔIEC mice. Arrows indicate the epithelial surface. (n=20) b, HMGB1 concentration in colonic mucus from HMGB1 WT and HMGB1 ΔIEC mice measured by ELISA. (n=6) c, Immunoblotting for HMGB1 in colonic mucus from HMGB1 WT and HMGB1 ΔIEC mice. (n=7) d, Immunostaining for HMGB1 (yellow) and Hoescht (blue) in Carnoy’s fixed proximal colon sections from SPF and GF C57BL/6 mice. Arrows indicate the epithelial surface. (n=6) e, Immunoblotting for HMGB1 in mucosal scrapings from SPF and GF C57BL/6 mice. (n=4) f, HMGB1 concentration in stool from SPF and GF C57BL/6 mice measured by ELISA. (n=6) Data are mean ± s.d. Significance determined by Student’s two-tailed T-tests. Each datapoint represents one individual mouse. Scale bars, 100 µm. Original magnification 400x

Article Snippet: Samples were analyzed using an HMGB1 detection kit per the manufacturer’s instructions (Chondrex).

Techniques: Immunostaining, Concentration Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: HMGB1 acts as an agent of host defense at the gut mucosal barrier

doi: 10.1101/2023.05.30.542477

Figure Lengend Snippet: Immunostaining of Carnoy’s fixed proximal colon sections from HMGB1 WT mice either using antibody treated with buffer (No peptide) or antibody incubated with the immunizing peptide (Peptide) to block HMGB1-specific staining. Scale bars, 100 µm. Original magnification 400x.

Article Snippet: Samples were analyzed using an HMGB1 detection kit per the manufacturer’s instructions (Chondrex).

Techniques: Immunostaining, Incubation, Blocking Assay, Staining

Journal: bioRxiv

Article Title: HMGB1 acts as an agent of host defense at the gut mucosal barrier

doi: 10.1101/2023.05.30.542477

Figure Lengend Snippet: a, Fluorescence in situ hybridization (FISH) using the EUB338 probe (purple) and Hoescht (blue) in Carnoy’s fixed proximal colon sections from HMGB1 WT and HMGB1 ΔIEC mice. Arrows indicate the epithelial surface. Dotted lines are placed at the epithelial surface and inner edge of the microbial community. Straight line indicates distance between host tissues and microbial community. (n=12) b, Distance measured between epithelium and bacterial cells in images represented in (a). Each datapoint is an average of 5 measurements for one individual mouse. c, Quantitative PCR for the bacterial 16S rRNA gene in 1 cm of colonic tissue from HMGB1 WT and HMGB1 ΔIEC mice. (n=6) d, Invasion of green fluorescent protein (GFP) labeled E. coli (SWW33) into mucus isolated from HMGB1 WT or HMGB1 ΔIEC mice. Original magnification 200x (n=3; 3 replicates) e, Percentage of the total GFP signal in mucus from HMGB1 WT vs. HMGB1 ΔIEC mice in images represented in (d). (n=3; 3 replicates) f, Appearance of GFP labeled E. coli (SWW33) exposed to buffer (control) or HMGB1. (n=3; 3 replicates) g, Flow cytometry of aggregates in samples of GFP labeled E. coli (SWW33) exposed to buffer (control) or HMGB1. (n=3; 3 replicates) h, Appearance of SYTO 9 labeled microbiota from C57BL/6 mice exposed to buffer (control) or HMGB1 labeled with AF647. Scale bars, 20 µm (n=3; 3 replicates) Data are mean ± s.d. Significance determined by Student’s two-tailed T-tests. Each datapoint represents one individual mouse. Scale bars, 100 µm and original magnification 400x, unless otherwise noted.

Article Snippet: Samples were analyzed using an HMGB1 detection kit per the manufacturer’s instructions (Chondrex).

Techniques: Fluorescence, In Situ Hybridization, Real-time Polymerase Chain Reaction, Labeling, Isolation, Control, Flow Cytometry, Two Tailed Test

Journal: bioRxiv

Article Title: HMGB1 acts as an agent of host defense at the gut mucosal barrier

doi: 10.1101/2023.05.30.542477

Figure Lengend Snippet: a, Quantitative reverse transcriptase (qRT) PCR for expression of Mucin1 (Muc1) , Mucin2 (Muc2) , and Mucin3 (Muc3) in colons from HMGB1 WT and HMGB1 ΔIEC mice. (n=9) b, Immunostaining for Mucin 2 (Muc2) in Carnoy’s fixed proximal colon sections from HMGB1 WT and HMGB1 ΔIEC mice. Arrows indicate the epithelial surface. (n=4) c, Lectin staining with fluorescently labeled Ulex europaeus agglutinin-1 (UEA-1) in Carnoy’s fixed proximal colon sections from HMGB1 WT and HMGB1 ΔIEC mice. Arrows indicate the epithelial surface. (n=4) Data are mean ± s.d. Significance determined by Student’s two-tailed T-tests. Each datapoint represents one individual mouse. Scale bars, 100 µm. Original magnification 400x.

Article Snippet: Samples were analyzed using an HMGB1 detection kit per the manufacturer’s instructions (Chondrex).

Techniques: Reverse Transcription, Quantitative RT-PCR, Expressing, Immunostaining, Staining, Labeling, Two Tailed Test

Journal: bioRxiv

Article Title: HMGB1 acts as an agent of host defense at the gut mucosal barrier

doi: 10.1101/2023.05.30.542477

Figure Lengend Snippet: a, Immunostaining for HMGB1 in Carnoy’s fixed proximal colon sections from HMGB1 WT mice. Focal plane optimized to capture gut microbes. Scale bars, 100 µm. Original magnification 400x. Arrows indicate the leading edge of gut microbes. (n=20) b, Shannon alpha-diversity index of ASV abundances using DNA isolated from mucosal scrapings of colons from HMGB1 WT and HMGB1 ΔIEC mice. (n=12 WT/18 ΔIEC) c, Canonical correspondence analysis (CCA) on ASV abundances using DNA isolated from mucosal scrapings of colons from HMGB1 WT and HMGB1 ΔIEC mice. (n=12 WT/18 ΔIEC) d, Dimensional reduction plots used to characterize microbiome differences between the indicated sites (stool and mucosal) in samples from HMGB1 WT and HMGB1 ΔIEC mice. (n=12 WT/18 ΔIEC) R 2 derived from permutational multivariate analysis of variance with site as the main variable. R 2 indicates the difference between the composition of the microbiota at the two sites in mice of each genotype (HMGB1 WT and HMGB1 ΔIEC ) and p-value indicates the significance of the difference in composition between the two sites. (n=12 WT/18 ΔIEC) e, Mean proportion of statistically different bacterial strains using DNA isolated from mucosal scrapings of colons from HMGB1 WT and HMGB1 ΔIEC mice. (n=12 WT/18 ΔIEC) Each datapoint represents one individual mouse except in (d) where each mouse has one datapoint for stool and one for mucosal sample.

Article Snippet: Samples were analyzed using an HMGB1 detection kit per the manufacturer’s instructions (Chondrex).

Techniques: Immunostaining, Isolation, Derivative Assay

Journal: bioRxiv

Article Title: HMGB1 acts as an agent of host defense at the gut mucosal barrier

doi: 10.1101/2023.05.30.542477

Figure Lengend Snippet: a, Amino acid sequence similarities among the known human HMGB1 target proteins Beclin-1 and Atg5 and bacterial FimH. The putative HMGB1 interaction motif was derived from the amino acid sequence similarities between Beclin-1 and Atg5 and common amino acid replacements. b,c, Flow cytometry of rHMGB1 binding to E. coli (BW25113) knocked out for FimH (ΔFimH) or ΔFimH E. coli complemented with plasmids encoding wild type FimH (ΔFimH WT ; TSETPRV) or FimH mutated in the conserved residues of the putative motif (ΔFimH MUT ; ASATARA). Both the percent E. coli positive for HMGB1 (b) and the amount of HMGB1 protein (mean fluorescence intensity (MFI)) bound to each bacterium (c) were assessed. (n=3; 3 replicates) d, Label transfer of Sulfo-SBED from HMGB1 to recombinant FimH lectin domain (FimH LD ). Recipient proteins were wild type FimH LD (WT; TSETPRV) or FimH LD mutated in the conserved amino acid residues (mutant; ASATARA) of the putative interaction motif. Transfer was assessed in the absence or presence of mannose. (3 replicates) e, E. coli colony forming units (CFU) adherent to Caco2 IEC as a percent of input. E. coli were treated with buffer, rHMGB1, or mannose prior to addition to the IEC. (n=6; 3 replicates) f, Percentage of FimH LD protein bound to a mannose coated plate in the presence of increasing amounts of rHMGB1. (n=3; 3 replicates) g, RBC agglutination by E. coli (SWW33) expressing wild type FimH (FimH:TSETPRV), knocked out for FimH (FimH: KO), or expressing FimH mutated in the ToH1 sequence (FimH:ASATARA and FimH:AAAAAAA). Numbers of bacteria decrease from left to right. (3 replicates) h, Immunoblotting for FimH in mucus isolated from HMGB1 WT and HMGB1 ΔIEC mice. (n=4) i, Quantification of band densitometry of immunoblots represented in (h). (n=4) j, Immunostaining for FimH (red) and Hoescht (blue) in Carnoy’s fixed proximal colon sections from HMGB1 WT and HMGB1 ΔIEC mice. Scale bars, 100 µm. Original magnification 400x. (n=15) k, Quantification of FimH positive bacteria in images represented in (j). l, Immunoblotting for FimH in E. coli (SWW33) exposed to increasing amounts of HMGB1. (3 replicates) m, PCR determination of the orientation of the DNA switch region govering Fim gene expression in E. coli (ΔFimE) treated with media conditioned by IEC organoids derived from HMGB1 ΔIEC mice (ΔIEC CM), HMGB1 WT mice (WT CM), or ΔIEC CM supplemented with rHMGB1. Phase-on denotes switch oriented toward production of Fim genes. ftsZ is used for normalization. n, Relative band density of phase-on in (l). Data are mean ± s.d. Significance determined by Student’s two-tailed T-tests for pairwise comparisons or one-way ANOVA with a Tukey post hoc test. Each datapoint represents one individual mouse.

Article Snippet: Samples were analyzed using an HMGB1 detection kit per the manufacturer’s instructions (Chondrex).

Techniques: Sequencing, Derivative Assay, Flow Cytometry, Binding Assay, Fluorescence, Recombinant, Mutagenesis, Agglutination, Expressing, Bacteria, Western Blot, Isolation, Immunostaining, Two Tailed Test

Journal: bioRxiv

Article Title: HMGB1 acts as an agent of host defense at the gut mucosal barrier

doi: 10.1101/2023.05.30.542477

Figure Lengend Snippet: a, Immunofluorescence staining of HMGB1 (red) bound to E. coli (BW25113) (green). Wild type E. coli (WT), E. coli knocked out for FimH (ΔFimH), or ΔFimH E. coli complemented with plasmids carrying either WT FimH (ΔFimH WT ) or FimH mutated in ToH1 (ΔFimH Mut ) exposed to rHMGB1 and SYTO 9 to label bacterial DNA. b, Flow cytometry for FimH expression on the surface of ΔFimH E. coli complemented with plasmids carrying either WT FimH (ΔFimH WT ) or FimH mutated in ToH1 (ΔFimH Mut ). c, Flow cytometry gating strategy for HMGB1 biding reported in d, Flow cytometry of rHMGB1 binding to E. coli of the indicated strains. e, Flow cytometry gating strategy for HMGB1 binding reported in (d). Data are mean ± s.d. Significance determined by Student’s two-tailed T-tests. Each datapoint represents one biological replicate.

Article Snippet: Samples were analyzed using an HMGB1 detection kit per the manufacturer’s instructions (Chondrex).

Techniques: Immunofluorescence, Staining, Flow Cytometry, Expressing, Binding Assay, Two Tailed Test

Journal: bioRxiv

Article Title: HMGB1 acts as an agent of host defense at the gut mucosal barrier

doi: 10.1101/2023.05.30.542477

Figure Lengend Snippet: a, Immunostaining for HMGB1 (yellow) and Hoescht (blue) in Carnoy’s fixed sections of resected colon from Non-IBD or UC patients. (n=16) b, Quantification of surface associated HMGB1 using images represented in (a). Staining intensity reported as relative fluorescent units (RFU) per μm 2 . (n=16) c, Surface associated HMGB1 reported in (b) graphed by inflammation severity. d, Immunostaining for FimH (red) and Hoescht (blue) in Carnoy’s fixed sections of resected colon from Non-IBD or UC patients. Serial tissue sections from the same patients represented in (a) (n=16) e, Quantification of FimH positive bacteria using images represented in (d). Reported as number of objects per high powered field. (n=16) f, Quantification of FimH positive bacteria reported in (e) graphed by inflammation severity. g, Surface HMGB1 and FimH positive bacteria plotted for each patient. The size of the closed circles corresponds to inflammation severity. Open ovals denote the population characteristics by group (non-IBD and UC). h, Two-way scatter plot of surface HMGB1 and FimH positive bacteria in each patient with a fitted curve. The relationship between HMGB1 and FimH was captured by non-linear regression. Data are mean ± s.d. Scale bars, 100 µm. Original magnification 400x. Each datapoint represents one individual person. Mann-Whitney U tests were used to compare HMGB1 and FimH in non-IBD vs. UC groups (two-group comparison), and Kruskal Wallis tests were used to assess the difference in HMGB1 and FimH among inflammation groups (three-group comparison).

Article Snippet: Samples were analyzed using an HMGB1 detection kit per the manufacturer’s instructions (Chondrex).

Techniques: Immunostaining, Staining, Bacteria, MANN-WHITNEY, Comparison

Journal: International immunology

Article Title: Inducible expression of nuclear factor IL-6 increases endogenous gene expression of macrophage inflammatory protein-1 alpha, osteopontin and CD14 in a monocytic leukemia cell line.

doi: 10.1093/intimm/10.12.1825

Figure Lengend Snippet: Fig. 1. Induction of lysozyme mRNA by stimulating NF-IL6 expression in M1 cells. (A) M1 cells stably transfected with an IPTG-inducible pOPRSVI-NFIL6 plasmid (NFIL6 M1 cells) were grown at 1.03105/ml and then 1 mM IPTG was added to the medium to stimulate NF-IL6 expression. Cell lysates collected at the indicated time points were tested by immunoblotting with anti-C/EBPβ polyclonal antibody. (B) Total RNA (15 µg/lane) was extracted from NFIL6 M1 cells at the indicated time points following stimulation with 1 mM IPTG and subjected to Northern blot hybridization with mouse lysozyme probe. The bottom panel shows a control hybridization with EF-1α-specific probe.

Article Snippet: After boiling for 5 min, cell lysates (13105 cells per lane) were separated by SDS–PAGE (4–20% gradient polyacrylamide gel), electrophoretically transferred to a nitrocellulose membrane and then analyzed for immunoreactivity with anti-C/EBPβ (NF-IL6) antibody (C-19; Santa Cruz Biotechnology, Santa Cruz, CA) by the enhanced chemiluminescence detection system (Dupont, Boston, MA).

Techniques: Expressing, Stable Transfection, Transfection, Plasmid Preparation, Western Blot, Northern Blot, Hybridization, Control

Journal: International immunology

Article Title: Inducible expression of nuclear factor IL-6 increases endogenous gene expression of macrophage inflammatory protein-1 alpha, osteopontin and CD14 in a monocytic leukemia cell line.

doi: 10.1093/intimm/10.12.1825

Figure Lengend Snippet: Fig. 3. NF-IL6 stimulates transcription of the MIP-1α promoter in transactivation assays. Murine MIP-1α reporter gene constructs (from top: MIP-1α promoter construct p-676Luc, p-341Luc, p-172Luc, p-99Luc, promoterless luciferase construct pGL3Basic) are shown schematically on the left. Numbers indicate positions relative to the transcription start site. The results of luciferase assays are illustrated on the right. NFIL6 M1 cells were co-transfected with 20 µg of these constructs and 2 µg of a sea-pansy luciferase vector (pRL-TK) as a transfection efficiency control. The cells were split into two equal parts post-transfection and cultured in the presence (1 mM) or absence of IPTG. The cultures were harvested 20 h after transfection and analyzed for luciferase activity. The promoter activity was determined as relative light units compared with the promoterless pGL3 basic vector. The data represent the mean 6 SD of three independent experiments.

Article Snippet: After boiling for 5 min, cell lysates (13105 cells per lane) were separated by SDS–PAGE (4–20% gradient polyacrylamide gel), electrophoretically transferred to a nitrocellulose membrane and then analyzed for immunoreactivity with anti-C/EBPβ (NF-IL6) antibody (C-19; Santa Cruz Biotechnology, Santa Cruz, CA) by the enhanced chemiluminescence detection system (Dupont, Boston, MA).

Techniques: Construct, Luciferase, Transfection, Plasmid Preparation, Control, Cell Culture, Activity Assay

Journal: International immunology

Article Title: Inducible expression of nuclear factor IL-6 increases endogenous gene expression of macrophage inflammatory protein-1 alpha, osteopontin and CD14 in a monocytic leukemia cell line.

doi: 10.1093/intimm/10.12.1825

Figure Lengend Snippet: Fig. 4. EMSA of the putative C/EBP binding sequences within the murine MIP-1α proximal promoter. (A) Nucleotide sequences of the murine MIP-1α proximal promoter. The transcriptional start site is overlined by a forward arrow and designated as position 11. The dashed underlines indicate putative C/EBP binding sites described previously (38). Sequences used to generate double-stranded oligonucleotides for EMSA studies are indicated by lines above the sequences, and designated A, B, C and D. Each oligonucleotide covers one C/EBP binding site. (B) The double-stranded A–D oligonucleotides were 32P-labeled and incubated with nuclear protein prepared from control NFIL6 M1 cells (lanes 1, 5, 9 and 13) or 12 h IPTG-treated NFIL6 M1 cells (lanes 2–4, 6–8, 10–12 and 14–16). The double-stranded competitor oligonucleotide from the human IL-6 promoter, which contains one NF-IL6 binding site, was added at a 50-fold molar excess over probe oligonucleotides (lanes 3, 7, 11 and 15). In lanes 4, 8, 12 and 16, rabbit polyclonal antibody to NF-IL6 was added to the binding reaction. The positions of free probe (F) and supershifted complex (S) are marked on the right side of each panel. The positions of DNA–nuclear protein complexes detected with probe A, B and D are also indicated on the right sides.

Article Snippet: After boiling for 5 min, cell lysates (13105 cells per lane) were separated by SDS–PAGE (4–20% gradient polyacrylamide gel), electrophoretically transferred to a nitrocellulose membrane and then analyzed for immunoreactivity with anti-C/EBPβ (NF-IL6) antibody (C-19; Santa Cruz Biotechnology, Santa Cruz, CA) by the enhanced chemiluminescence detection system (Dupont, Boston, MA).

Techniques: Binding Assay, Labeling, Incubation, Control

Journal: International immunology

Article Title: Inducible expression of nuclear factor IL-6 increases endogenous gene expression of macrophage inflammatory protein-1 alpha, osteopontin and CD14 in a monocytic leukemia cell line.

doi: 10.1093/intimm/10.12.1825

Figure Lengend Snippet: Fig. 5. Transactivation of MIP-1α promoter by NF-IL6 requires two NF-IL6 binding sites. (A) Competition analysis of the mutated NF-IL6 binding sites using an EMSA. The double-stranded B and D oligonucleotides were 32P labeled and incubated with nuclear protein prepared from control NFIL6 M1 cells (lanes 1 and 5) or 12 h IPTG-treated NFIL6 M1 cells (lanes 2–4 and 6–8). A 50-fold molar excess of unlabeled wild- type oligonucleotides B or D (lanes 3 and 7) and NF-IL6 binding site mutant oligonucleotides mB or mD (lanes 4 and 8) were added to the binding reaction. Symbols marked on the right side of each panel are as described in the legend to Fig. 4. (B) Murine MIP-1α reporter gene constructs (from top: MIP-1α promoter construct p-172Luc, position –115 to –114 NF-IL6 binding site mutated construct pMutBLuc and position –54 to –53 NF-IL6 binding site mutated construct pMutDLuc, promoterless luciferase construct pGL3Basic) are shown schematically on the left. Numbers indicate positions relative to the transcription start site. The closed circles B and D represent NF-IL6 binding sites located within oligonucleotides B and D used in EMSA respectively. The results of luciferase assays are illustrated on the right. NFIL6 M1 cells were co-transfected with 20 µg of these constructs and 2 µg of a sea-pansy luciferase vector (pRL-TK) as a transfection efficiency control. The cells were split into two equal parts post-transfection and cultured in the presence (1 mM) or absence of IPTG. The cultures were harvested 20 h after transfection and analyzed for luciferase activity. The promoter activity was determined as relative light units compared with the promoterless pGL3 basic vector. The data represent the mean 6 SD of three independent experiments.

Article Snippet: After boiling for 5 min, cell lysates (13105 cells per lane) were separated by SDS–PAGE (4–20% gradient polyacrylamide gel), electrophoretically transferred to a nitrocellulose membrane and then analyzed for immunoreactivity with anti-C/EBPβ (NF-IL6) antibody (C-19; Santa Cruz Biotechnology, Santa Cruz, CA) by the enhanced chemiluminescence detection system (Dupont, Boston, MA).

Techniques: Binding Assay, Labeling, Incubation, Control, Mutagenesis, Construct, Luciferase, Transfection, Plasmid Preparation, Cell Culture, Activity Assay

Journal: Scientific Reports

Article Title: Dynamics of clusterin protein expression in the brain and plasma following experimental traumatic brain injury

doi: 10.1038/s41598-019-56683-6

Figure Lengend Snippet: Temporo-spatial evolution of prolonged clusterin immunoreactivity in the cortex, dentate gyrus, and thalamus ipsilateral to the injury. (A) A brightfield photomicrograph of a Nissl-stained coronal section from a rat perfused for immunohistochemistry at 1 month after TBI. The lesion core is indicated by an arrow. Dashed boxes indicate the brain regions with prominent clusterin immunoreactivity (ir) in layer IV of the ipsilateral cortex (Cx; panels B,E,H,K), molecular layer of the dentate gyrus (DG; panels C,F,I,L), and dorsal aspect of the thalamus (Th; panels D,G,J,M). Higher-power darkfield photomicrographs show that at 7 d post-TBI, punctate clusterin immunoreactivity (arrowheads) was prominently present in (B) layer IV of the perilesional cortex, (C) in the molecular layer of ipsilateral dentate gyrus, and faintly in (D) the ipsilateral dorsal thalamus. At 14 d post-TBI, (G) the thalamic immunostaining increased. Cortical (E,H) and hippocampal (F,I) staining persisted prominently for up to 1 month post-TBI, becoming substantially weaker by ( cortex K , hippocampus L) 12 months post-TBI. In contrast, (J,M) thalamic staining remained prominent from 1 month until 12 months post-TBI. No immunostaining was observed in the corresponding contralateral brain areas. Abbreviations: CA3, cornu Ammonis 3; d, days; DLG, dorsal lateral geniculate nucleus; G, granule cell layer of the dentate gyrus; H, hilus; LDVL, laterodorsal thalamic nucleus, ventrolateral part; M, molecular layer of the dentate gyrus; mo, months; Rt, reticular thalamic nucleus; TBI, traumatic brain injury; VPL, ventral posterolateral thalamic nucleus; VPM, ventral posteromedial thalamic nucleus. Scale bar equals 100 µm for all panels.

Article Snippet: In cohort 1, primary antibody incubation was performed with a rabbit polyclonal antibody raised against clusterin (1:1000, #STJ92346, St John’s Laboratory, London, UK).

Techniques: Staining, Immunohistochemistry, Immunostaining

Journal: Scientific Reports

Article Title: Dynamics of clusterin protein expression in the brain and plasma following experimental traumatic brain injury

doi: 10.1038/s41598-019-56683-6

Figure Lengend Snippet: Post-TBI expression of clusterin is prominent in the extracellular space. Double-immunofluorescence revealed that in the perilesional cortex, ipsilateral dentate gyrus, and ipsilateral thalamus, clusterin-immunoreactivity did not colocalise with the (A–C) neuronal marker NeuN, (D–F) astrocyte marker GFAP, microglial markers (G–I) CD68 or (J–L) OX42, or (M–O) the mitochondrial marker MT-CO1. In the ipsilateral dorsal thalamus, robust clusterin immunoreactivity surrounded the cells labelled with microglial markers (I) CD68 and (L) OX42, and (O) mitochondria labelled with MT-CO1. All images were taken from rats perfused at 1 month post-TBI. Abbreviations: CD68, cluster of differentiation 68; Clu, clusterin; GFAP, glial fibrillary acidic protein; MT-CO1, mitochondrially encoded cytochrome c oxidase 1; NeuN, neuronal nuclei; OX42, antibody against CD11b/c; Scale bar = 50 µm for all panels.

Article Snippet: In cohort 1, primary antibody incubation was performed with a rabbit polyclonal antibody raised against clusterin (1:1000, #STJ92346, St John’s Laboratory, London, UK).

Techniques: Expressing, Immunofluorescence, Marker

Journal: Scientific Reports

Article Title: Dynamics of clusterin protein expression in the brain and plasma following experimental traumatic brain injury

doi: 10.1038/s41598-019-56683-6

Figure Lengend Snippet: Elevated Clu mRNA expression was observed in the brain at 3 months post-TBI. TaqMan RT-qPCR analysis revealed increased Clu mRNA expression in the (A) perilesional cortex (FC 3.3, p < 0.01) and (B) ipsilateral thalamus (FC 2.4, p < 0.05) of the rats at 3 months post-TBI as compared to sham-operated controls (n = 9 TBI, 6 sham; each dot in panels A-B refers to one animal). Clusterin Ct values were normalised to the housekeeping gene GAPDH. Statistical significances: *p < 0.05; **p < 0.01 (Mann-Whitney U test). Abbreviations: Clu, clusterin; Ct, cycle threshold; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; sham, sham-operated controls.

Article Snippet: In cohort 1, primary antibody incubation was performed with a rabbit polyclonal antibody raised against clusterin (1:1000, #STJ92346, St John’s Laboratory, London, UK).

Techniques: Expressing, Quantitative RT-PCR, MANN-WHITNEY, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: Dynamics of clusterin protein expression in the brain and plasma following experimental traumatic brain injury

doi: 10.1038/s41598-019-56683-6

Figure Lengend Snippet: Acute reduction in plasma clusterin levels after TBI. ( A ) The dilution curve demonstrated linearity, indicating no significant matrix interference effect in the ELISA assay. (B) Clusterin levels in plasma derived from the cardiac puncture at very acute post-TBI time-points (<6 h, i.e ., 2 h-6 h post-TBI) were lower than that in controls (15%, p < 0.01) or that at 6 months post-TBI (21%, p < 0.01). At the other time-points, there was no difference between the TBI and control animals, or within the TBI groups. Moreover, at 6 months post-TBI, the plasma clusterin levels did not distinguish the rats with (open circles) or without (filled circles) spontaneous seizures (p > 0.05). (C) ROC analysis indicated that plasma clusterin levels sampled <6 h from TBI distinguished the animals with TBI from controls with an AUC of 0.851 (p < 0.05) and (D) from the 6 months post-TBI group with an AUC of 0.917 (p < 0.01). Statistical significances: **p < 0.01 compared with the control group; ##p < 0.01 compared to 6 months post-TBI (Mann-Whitney U test). Abbreviations: AUC, area under curve; C, controls; d, days; h, hours; mo, months; R 2 , coefficient of determination; wk, weeks.

Article Snippet: In cohort 1, primary antibody incubation was performed with a rabbit polyclonal antibody raised against clusterin (1:1000, #STJ92346, St John’s Laboratory, London, UK).

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Derivative Assay, Control, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Dynamics of clusterin protein expression in the brain and plasma following experimental traumatic brain injury

doi: 10.1038/s41598-019-56683-6

Figure Lengend Snippet: Study design for clusterin analysis in brain tissue, plasma, and CSF. Spatiotemporal expression of clusterin protein in the brain after TBI was investigated in cohorts 1–2. Chronic expression of clusterin mRNA in the brain was assessed using RT-qPCR in cohort 3. Post-TBI plasma clusterin levels were assessed using ELISA in cohorts 4–5. In cohort 5, 5 of the 16 TBI rats had epilepsy. Consequently, the study was powered to diagnose post-traumatic epilepsy in the TBI group if the AUC was ≥0.900. Finally, clusterin levels in CSF was analysed using iTRAQ proteomics in the chronic cohort 2. Abbreviations: CSF, cerebrospinal fluid; d, days; ELISA, enzyme-linked immunosorbent assay; h, hours; IHC, immunohistochemistry; iTRAQ, isobaric tag for relative and absolute quantification; RNA-Seq, RNA sequencing; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; sham, sham-operated controls; TBI, traumatic brain injury.

Article Snippet: In cohort 1, primary antibody incubation was performed with a rabbit polyclonal antibody raised against clusterin (1:1000, #STJ92346, St John’s Laboratory, London, UK).

Techniques: Clinical Proteomics, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Multiplex sample analysis, Immunohistochemistry, Quantitative Proteomics, RNA Sequencing, Reverse Transcription, Real-time Polymerase Chain Reaction