recombinant s100b protein  (TargetMol)

Journal: Journal of Advanced Research

Article Title: S100B induces angiogenesis via the clathrin/FOXO1/β-catenin signaling pathway and contributes to Bevacizumab resistance in epithelial ovarian cancer

doi: 10.1016/j.jare.2025.05.060

Figure Lengend Snippet: Flowchart of the study. First, BEV-sensitive and BEV-resistant ovarian cancer mouse models were constructed, and RNA sequencing was performed on tumor tissue. Then, a BEV-related prognostic signature was established using machine learning, and S100B was identified as the most important molecule regulating BEV sensitivity in ovarian cancer. Its function and mechanism were analyzed in vitro. Finally, BEV efficacy when combined with an S100B inhibitor was verified in vivo. BEV: Bevacizumab; DEGs: Differentially expressed genes.

Article Snippet: During phenotype experiments with HUVECs, recombinant S100B protein (MCE, HY- P70659 ) was added at concentrations of 10, 100, and 1000 mg/mL.

Techniques: Construct, RNA Sequencing, In Vitro, In Vivo

Journal: Journal of Advanced Research

Article Title: S100B induces angiogenesis via the clathrin/FOXO1/β-catenin signaling pathway and contributes to Bevacizumab resistance in epithelial ovarian cancer

doi: 10.1016/j.jare.2025.05.060

Figure Lengend Snippet: Screening S100B as the main molecule regulating the sensitivity of ovarian cancer to BEV. (A) Kaplan–Meier survival analysis of OS (left) and PFS (right) of patients with high S100B expression treated with chemotherapy with or without BEV (from the GSE140082 dataset). (B) Kaplan–Meier survival analysis of OS (left) and PFS (right) of patients with low S100B expression treated with chemotherapy with or without BEV (from the GSE140082 dataset). (C) Western blot analysis of S100B protein expression in tumor tissue from BEV-sensitive and BEV-resistant mice. (D) Western blot analysis of S100B protein levels in mouse tumor tissue. (E) Immunohistochemical staining of S100B protein in BEV-sensitive and BEV-resistant mice. (F) Statistical histochemical staining of S100B protein in mouse tumor tissue. BEV:Bevacizumab; **: P < 0.01.

Article Snippet: During phenotype experiments with HUVECs, recombinant S100B protein (MCE, HY- P70659 ) was added at concentrations of 10, 100, and 1000 mg/mL.

Techniques: Expressing, Western Blot, Immunohistochemical staining, Staining

Journal: Journal of Advanced Research

Article Title: S100B induces angiogenesis via the clathrin/FOXO1/β-catenin signaling pathway and contributes to Bevacizumab resistance in epithelial ovarian cancer

doi: 10.1016/j.jare.2025.05.060

Figure Lengend Snippet: Co-culture of S100B overexpressing ovarian cancer cells promotes HUEVC angiogenesis and migration. (A) Representative Western blot images and their densitometric quantification showing comparative S100B protein expression profiles among the three ovarian cancer cell lines (A2780, HEY, and OVCAR3) (B) Western blot analysis and statistical analysis of OVCAR3 and HEY cells overexpressing S100B. RT-qPCR analysis of alternative angiogenic factor mRNA expression levels in (C) OVCAR3 and (D) HEY ovarian cancer cell lines following S100B overexpression. (E) The level of S100B in the supernatant of OVCAR3 and HEY cells overexpressing S100B significantly increased, as determined by ELISA. (F) There was no significant change in VEGFA levels in the supernatant of OVCAR3 and HEY cells overexpressing S100B. (G, H) HUVEC tube formation significantly increased after co-culture with ovarian cancer cell lines overexpressing S100B, with or without exogenous BEV. (I, J) Co-culture with ovarian cancer cells overexpressing S100B significantly promoted HUVEC sprouting with or without exogenous BEV. (K, L) Co-culture of ovarian cancer cells overexpressing S100B significantly promoted migration of HUVECs in a Transwell system with or without exogenous BEV. (M, N) Co-culture of ovarian cancer cell lines overexpressing S100B significantly promoted healing of HUVECs with or without exogenous BEV in a scratch assay. BEV: Bevacizumab; NC: negative control; OE: overexpression; *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001. Scale: All panels are 50 μm.

Article Snippet: During phenotype experiments with HUVECs, recombinant S100B protein (MCE, HY- P70659 ) was added at concentrations of 10, 100, and 1000 mg/mL.

Techniques: Co-Culture Assay, Migration, Western Blot, Expressing, Quantitative RT-PCR, Over Expression, Enzyme-linked Immunosorbent Assay, Wound Healing Assay, Negative Control

Journal: Journal of Advanced Research

Article Title: S100B induces angiogenesis via the clathrin/FOXO1/β-catenin signaling pathway and contributes to Bevacizumab resistance in epithelial ovarian cancer

doi: 10.1016/j.jare.2025.05.060

Figure Lengend Snippet: Exogenous recombinant S100B protein promoted vascular formation and migration of HUEVCs. Different concentrations of recombinant S100B protein, with or without BEV, promoted (A, B) angiogenesis in a tube formation assay, (C, D) sprouting in a fibrin bead sprouting assay, (E, F) migration in a Transwell assay, and (G, H) healing of endothelial cells in a wound healing assay. (I) Tip cell marker expression in HUVECs significantly increased by rt-qPCR detection after treatment with recombinant S100B protein. BEV: Bevacizumab; *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001. Scale: All panels are 50 μm.

Article Snippet: During phenotype experiments with HUVECs, recombinant S100B protein (MCE, HY- P70659 ) was added at concentrations of 10, 100, and 1000 mg/mL.

Techniques: Recombinant, Migration, Tube Formation Assay, Transwell Assay, Wound Healing Assay, Marker, Expressing, Quantitative RT-PCR

Journal: Journal of Advanced Research

Article Title: S100B induces angiogenesis via the clathrin/FOXO1/β-catenin signaling pathway and contributes to Bevacizumab resistance in epithelial ovarian cancer

doi: 10.1016/j.jare.2025.05.060

Figure Lengend Snippet: S100B enters endothelial cells through clathrin-dependent endocytosis to promote angiogenesis and migration. (A) Western blot detection showed that there was no significant change in RAGE receptor expression in endothelial cells after exogenous S100B treatment. (B) Western blot detection showed that protein levels of S100B in endothelial cells significantly increased after co-culture with ovarian cancer cells overexpressing S100B. (C) S100B total protein and nuclear protein in endothelial cells significantly increased after treatment with exogenous S100B, as determined by western blot analysis. (D) Western blot analysis of protein levels of S100B in endothelial cells after pretreatment with the clathrin-mediated endocytosis inhibitor Pitstop-2 or caveolae/caveolin-1-mediated endocytosis inhibitor nystatin. Changes in the (F, G) tubular phenotype, (H, I) sprouting phenotype, (J, K) migration phenotype (Transwell assay), and (L–M) wound healing phenotype (scratch migration assay) of endothelial cells after treatment with the RAGE receptor inhibitor FPS-ZM1, the clathrin-mediated endocytosis inhibitor Pitstop-2, and the caveolae/caveolin-mediated endocytosis inhibitor nystatin. BEV: Bevacizumab; *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001. Scale: All panels are 50 μm.

Article Snippet: During phenotype experiments with HUVECs, recombinant S100B protein (MCE, HY- P70659 ) was added at concentrations of 10, 100, and 1000 mg/mL.

Techniques: Migration, Western Blot, Expressing, Co-Culture Assay, Transwell Assay

Journal: Journal of Advanced Research

Article Title: S100B induces angiogenesis via the clathrin/FOXO1/β-catenin signaling pathway and contributes to Bevacizumab resistance in epithelial ovarian cancer

doi: 10.1016/j.jare.2025.05.060

Figure Lengend Snippet: S100B promotes tube formation and migration of endothelial cells through the FOXO1/β-catenin signaling pathway. (A) DIA proteomics thermogram of differentially expressed proteins in HUVECs with or without exogenous S100B. (B) Function and pathway enrichment analysis by Metascape database of differentially expressed proteins. (C–E) Western blot analysis showed that levels of S100B in ovarian cancer tissue of BEV-resistant mice significantly increased, while FOXO1 levels decreased. (F) Histogram of an RT-qPCR analysis of downstream transcriptional target genes of β-catenin. (G–J) Western blot analysis showed that FOXO1 total protein and nuclear protein levels in endothelial cells significantly decreased after treatment with exogenous S100B, while levels of β-catenin and MMP7 significantly increased. (K–M) Western blot analysis of FOXO1 and β-catenin levels in S100B-treated endothelial cells overexpressing FOXO1 compared with the control group. Endothelial cells transfected with a control virus or FOXO1 -overexpressing lentivirus and treated with S100B were evaluated for their (N) tubular phenotype by tube formation assay, (O) sprouting phenotype by fibrin bead sprouting assay, and (P) migration phenotype by Tranwell assay. (Q) Schematic diagram of the mechanism of exogenous S100B on endothelial cell angiogenesis. *: P < 0.05; **: P < 0.01;^: P < 0.05 vs. S100B−/oeFOXO1−; #: P < 0.05 vs. S100B−/oeFOXO1+; &: P < 0.05 vs. S100B+/oeFOXO1 − . Scale: All panels are 50 μm.

Article Snippet: During phenotype experiments with HUVECs, recombinant S100B protein (MCE, HY- P70659 ) was added at concentrations of 10, 100, and 1000 mg/mL.

Techniques: Migration, Western Blot, Quantitative RT-PCR, Control, Transfection, Virus, Tube Formation Assay

Journal: Journal of Advanced Research

Article Title: S100B induces angiogenesis via the clathrin/FOXO1/β-catenin signaling pathway and contributes to Bevacizumab resistance in epithelial ovarian cancer

doi: 10.1016/j.jare.2025.05.060

Figure Lengend Snippet: The S100B inhibitor pentamidine in combination with BEV improves the ovarian cancer response compared to BEV alone. (A) Flowchart of the in vivo experiment. Two weeks after intraperitoneal inoculation of ovarian cancer cells, drug treatment was administered. The mice were randomly divided into a normal saline treatment control group, BEV treatment group, pentamidine treatment group, and BEV with pentamidine treatment group. (B) Line chart of the tumor fluorescence intensity of the four groups of mice. (C) Tumor fluorescence imaging of mice after tumor formation (week 2), after killing the control group and pentamidine treatment group (week 4–6), after killing the BEV treatment group (week 8–9), and after killing the BEV with pentamidine treatment group (week 13–14). (D) Kaplan–Meier survival curves of the four groups of mice. (E) CD31 immunohistochemical staining and (F) MVD statistics of tumor tissue from the four groups of mice. (E) S100B immunohistochemical staining and S100B (G) staining intensity of tumor tissue from the four groups of mice. BEV: Bevacizumab; MVD: Micro-vessel density; *: P < 0.05; **: P < 0.01;^: P < 0.05 vs. NC; #: P < 0.05 vs. pentamidine; &: P < 0.05 vs. BEV.

Article Snippet: During phenotype experiments with HUVECs, recombinant S100B protein (MCE, HY- P70659 ) was added at concentrations of 10, 100, and 1000 mg/mL.

Techniques: In Vivo, Saline, Control, Fluorescence, Imaging, Immunohistochemical staining, Staining

Journal: PLOS Biology

Article Title: The suprachiasmatic nucleus regulates brown fat thermogenesis in male mice through an adrenergic receptor ADRB3-S100B signaling pathway

doi: 10.1371/journal.pbio.3003534

Figure Lengend Snippet: (A) Volcano plot showing differentially expressed genes (DEGs) in interscapular BAT from sham and SCNx mice under TRF-STE. DEGs were defined by fold change > 2, p < 0.01. Commonly upregulated genes are highlighted in red and downregulated genes in green at both ZT4 and ZT16. n = 3 per group. (B) IPA of DEGs at ZT4 and ZT16. Pathways with significant enrichment ( p < 0.01) are shown, with activation z-scores indicated for each pathway. (C) DAVID pathway analysis of S100 family-associated DEGs. Enriched KEGG pathways are shown in yellow, and Gene Ontology biological processes (BP) are shown in gray. (D and E) Correlation analysis of RNA-seq-derived S100b expression with cell proliferation markers ( D ) and senescence-related genes ( E ) in sham and SCNx mice. Pearson correlation coefficients and p -values are shown. (F and G) Cell proliferation analysis via EdU incorporation. Representative images showing EdU incorporation in interscapular BAT from sham and SCNx mice at ZT4 and ZT16 under Ad-STE and TRE-STE with 4°C and 30°C as control (F) . Quantification of EdU + nuclei as a percentage of total (Hoechst-stained) nuclei (G) . n = 5 per group. (H–K) Assessment of S100B-induced proliferation in PDGFRα + preadipocytes. PDGFRα-positive stromal vascular fraction (SVF) cells (H) , representative images of EdU-positive cells treated with recombinant S100B (I) , statistical analysis of EdU + nuclei (J) , and cell growth curves of control vs. S100B-treated preadipocytes (K) . (L) Expression of differentiation-related genes during SVF from interscapular BAT induced to differentiate into mature adipocytes. n = 6 per group. (M) Representative β-galactosidase staining images showing senescent cell abundance in interscapular BAT from sham and SCNx mice under TRF-STE. Data are presented as mean ± SD. Statistical significance was determined using unpaired two-tailed Student t test ( G , J, and L ), Pearson correlation analysis ( D and E ), and two-way ANOVA with Sidak’S multiple comparisons test (K) . * p < 0.05, ** p < 0.01 and **** p < 0.0001. Scale bars, 100 μm (F and M) , 50 μm (I) , 20 μm ( H and magnified view of I ). The data underlying the graphs shown in the figure can be found in

Article Snippet: For EdU-labeled cell proliferation of SVF, 0.1 μM recombinant S100B protein (TMPJ-00990, Targetmol USA) or saline and 10 μM EdU were added to the medium.

Techniques: Activation Assay, RNA Sequencing, Derivative Assay, Expressing, Control, Staining, Recombinant, Two Tailed Test

Journal: PLOS Biology

Article Title: The suprachiasmatic nucleus regulates brown fat thermogenesis in male mice through an adrenergic receptor ADRB3-S100B signaling pathway

doi: 10.1371/journal.pbio.3003534

Figure Lengend Snippet: (A) S100b expression levels under various nutritional states (fasting, HFD, and TRF) and environmental temperatures (4°C, 21°C, and 30°C). n = 4 per group. (B) S100b and Ucp1 expression levels in indicated temperatures. n = 4 per group. (C) Serum S100B levels were measured by ELISA in sham and SCNx mice under Ad-STE and TRF-STE at ZT4 and ZT16. n = 4 per group. (D and E) Serum biochemical assays showing levels of TG ( D ) and NEFA ( E ) in sham and SCNx mice under Ad-STE ( n = 6 per group). (F) Representative immunohistochemistry images of S100B protein in interscapular BAT from sham and SCNx mice under Ad-STE and TRF-STE conditions. Scale bars, 50μm. (G and H) Western blot analysis ( G ) and densitometric quantification ( H ) of S100B, p21, and CCND1 protein levels in interscapular BAT from sham and SCNx mice at ZT4 and ZT16. n = 4 per group. Red arrows indicate quantified bands. (I) Relative mRNA levels of indicated genes in interscapular BAT from sham and SCNx BAT following ADRB3 antagonist with SR59230A. n = 6 per group. SR: SR59230A. (J and K) Structural prediction of the human ADRB3-S100B complex using AlphaFold2/ColabFold. The human ADRB3/S100B complex model ( J ) was predicted by ColabFold and colored in blue, cyan, yellow, and orange according to different prediction confidence (pLDDT, predicted local distance difference test). Surface electrostatic representation showing ADRB3 (deep teal) and S100B (orange) with positive and negative charges indicated in blue and red, respectively (K) . ( L ) coIP of ADRB3 and S100B in HEK293T cells. (M and N) Western blot ( M ) and densitometry analysis ( N ) of ADRB3 protein levels in interscapular BAT following S100b knockdown or overexpression in vivo. n = 4 per group. Red arrows indicate quantified bands. (O and P) ECAR analysis ( O ) and quantification of basal glycolysis and glycolytic capacity ( P ) in primary preadipocytes treated with S100B and/or β3-agonist CL-316243. Glucose, oligomycin, and 2-DG were sequentially injected. Data are presented as mean ± SEM. Control, CL, 0.1 µM S100B and 0.2 µM S100B: n = 6; 0.1 µM S100B + CL and 0.2 µM S100B+CL: n = 3. (Q) Schematic model illustrating the proposed ADRB3-S100B signaling axis. SCN lesioning enhances SNS activity, thereby promoting ADRB3 signaling and upregulating S100B expression. S100B, in turn, increases ADRB3 sensitivity, establishing a positive feedback loop that sustains thermogenesis and stimulates preadipocyte proliferation. Created in BioRender.com. Unless otherwise indicated, data are presented as mean ± SD. NS: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 by unpaired two-tailed Student t test ( A – E , H , I , N, and P ). The data underlying the graphs shown in the figure can be found in S1 Source Data. Raw blot images can be found in S1 Raw Images.

Article Snippet: For EdU-labeled cell proliferation of SVF, 0.1 μM recombinant S100B protein (TMPJ-00990, Targetmol USA) or saline and 10 μM EdU were added to the medium.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Western Blot, Structural Proteomics, Knockdown, Over Expression, In Vivo, Injection, Control, Activity Assay, Two Tailed Test

Journal: PLOS Biology

Article Title: The suprachiasmatic nucleus regulates brown fat thermogenesis in male mice through an adrenergic receptor ADRB3-S100B signaling pathway

doi: 10.1371/journal.pbio.3003534

Figure Lengend Snippet: (A) Schematic of AAV-mediated S100b knockdown or scramble control delivery into interscapular BAT of SCNx mice. (B) Relative mRNA expression of the indicated genes in interscapular BAT from SCNx mice under TRF-STE with scramble or S100b targeting shRNA. n = 6 per group. (C and D) Western blot analysis of S100B and p21 ( C ) and corresponding densitometry quantification ( D ) in interscapular BAT from SCNx mice under TRF-STE ( n = 4 per group). (E and F) EdU incorporation analysis in interscapular BAT from SCNx mice following S100b knockdown or scramble control. Representative images ( E ) and quantification of EdU-positive nuclei relative to total Hoechst-positive nuclei (F) . (G) Body temperature profiles of SCNx mice with S100b knockdown or scramble control under Ad-STE and TRF-STE (scramble: n = 6, S100b knockdown: n = 5). Data presented as mean ± SEM. Corresponding interscapular BAT and tail temperature data are shown in . (H) Schematic illustrating AAV-mediated overexpression of S100b (AAV-DIO- S100b + AAV-CAG-Cre) or EGFP control in interscapular BAT of WT mice. (I) Relative mRNA levels of indicated genes in interscapular BAT from EGFP or S100b overexpressing mice under TRF-STE ( n = 6 per group). (J and K) Western blot analysis of S100B and p21 proteins ( J ) and corresponding densitometry ( K ) from interscapular BAT of EGFP or S100b -overexpressing mice ( n = 4 per group). (L and M) EdU incorporation analysis in interscapular BAT from EGFP and S100b -overexpressing mice. Representative images ( L ) and quantification of EdU + cells (M) . n = 6 per group. (N) Body temperature responses under Ad-STE and TRF-STE in WT mice with EGFP or S100b overexpression in interscapular BAT. Data presented as mean ± SEM. n = 6 per group. See for interscapular BAT and tail temperature profiles. Unless otherwise indicated, data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. Significance determined by unpaired two-tailed Student t test ( B , D , F , I , K, and M ) or two-way ANOVA with Sidak’S multiple comparisons test (G, N) . Scale bars, 50 μm (E, L) , 20 μm (magnified view of E , L ). Schematic ( A , H ) created in BioRender.com. The data underlying the graphs shown in the figure can be found in S1 Source Data. Raw blot images can be found in S1 Raw Images.

Article Snippet: For EdU-labeled cell proliferation of SVF, 0.1 μM recombinant S100B protein (TMPJ-00990, Targetmol USA) or saline and 10 μM EdU were added to the medium.

Techniques: Knockdown, Control, Expressing, shRNA, Western Blot, Over Expression, Two Tailed Test

Journal: PLOS Biology

Article Title: The suprachiasmatic nucleus regulates brown fat thermogenesis in male mice through an adrenergic receptor ADRB3-S100B signaling pathway

doi: 10.1371/journal.pbio.3003534

Figure Lengend Snippet: (A) LL exposure paradigm is used to disrupt SCN rhythmicity. Arrhythmic mice were selected for subsequent analysis . (B) Relative mRNA expression of indicated genes in interscapular BAT from WT mice under light/dark cycle (LD) or LL during TRF-STE ( n = 4 per group). (C and D) Western blot analysis ( C ) and densitometry ( D ) of S100B and p21 proteins in interscapular BAT from mice under LD or LL conditions ( n = 4 per group). (E and F) EdU staining in interscapular BAT from mice under LD or LL. Representative images ( E ) and quantification of EdU + cells ( F ) normalized to Hoechst staining. (G) Body temperature profiles of WT mice under LD or LL conditions during TRF-STE. Data presented as mean ± SEM. n = 4 per group. See for additional temperature metrics. (H) Working model illustrating the SCN-ADRB3-S100B axis in BAT during TRF conducted at ZT16-ZT20 in a subthermoneutral environment, the SCN regulates BAT thermogenic plasticity via SNS output. SCN lesioning enhances ADRB3 signaling and S100B expression, which together form a positive feedback loop that amplifies β3-adrenergic sensitivity, promotes preadipocyte proliferation, suppresses senescence, and sustains glucose-driven thermogenesis. Unless otherwise indicated, data are presented as mean ± SD. * p < 0.05, ** p < 0.01 and **** p < 0.0001. Significance determined by unpaired two-tailed Student t test ( B , D, and F ) or two-way ANOVA with Sidak’S multiple comparisons test (G) . Scale bars, 50 μm (E) , 20 μm (magnified view of E ). Schematic ( A ) and working model ( H ) created in BioRender.com. The data underlying the graphs shown in the figure can be found in S1 Source Data. Raw blot images can be found in S1 Raw Images.

Article Snippet: For EdU-labeled cell proliferation of SVF, 0.1 μM recombinant S100B protein (TMPJ-00990, Targetmol USA) or saline and 10 μM EdU were added to the medium.

Techniques: Expressing, Western Blot, Staining, Two Tailed Test

Journal: PLOS Biology

Article Title: The suprachiasmatic nucleus regulates brown fat thermogenesis in male mice through an adrenergic receptor ADRB3-S100B signaling pathway

doi: 10.1371/journal.pbio.3003534

Figure Lengend Snippet: (A) Volcano plot showing differentially expressed genes (DEGs) in interscapular BAT from sham and SCNx mice under TRF-STE. DEGs were defined by fold change > 2, p < 0.01. Commonly upregulated genes are highlighted in red and downregulated genes in green at both ZT4 and ZT16. n = 3 per group. (B) IPA of DEGs at ZT4 and ZT16. Pathways with significant enrichment ( p < 0.01) are shown, with activation z-scores indicated for each pathway. (C) DAVID pathway analysis of S100 family-associated DEGs. Enriched KEGG pathways are shown in yellow, and Gene Ontology biological processes (BP) are shown in gray. (D and E) Correlation analysis of RNA-seq-derived S100b expression with cell proliferation markers ( D ) and senescence-related genes ( E ) in sham and SCNx mice. Pearson correlation coefficients and p -values are shown. (F and G) Cell proliferation analysis via EdU incorporation. Representative images showing EdU incorporation in interscapular BAT from sham and SCNx mice at ZT4 and ZT16 under Ad-STE and TRE-STE with 4°C and 30°C as control (F) . Quantification of EdU + nuclei as a percentage of total (Hoechst-stained) nuclei (G) . n = 5 per group. (H–K) Assessment of S100B-induced proliferation in PDGFRα + preadipocytes. PDGFRα-positive stromal vascular fraction (SVF) cells (H) , representative images of EdU-positive cells treated with recombinant S100B (I) , statistical analysis of EdU + nuclei (J) , and cell growth curves of control vs. S100B-treated preadipocytes (K) . (L) Expression of differentiation-related genes during SVF from interscapular BAT induced to differentiate into mature adipocytes. n = 6 per group. (M) Representative β-galactosidase staining images showing senescent cell abundance in interscapular BAT from sham and SCNx mice under TRF-STE. Data are presented as mean ± SD. Statistical significance was determined using unpaired two-tailed Student t test ( G , J, and L ), Pearson correlation analysis ( D and E ), and two-way ANOVA with Sidak’S multiple comparisons test (K) . * p < 0.05, ** p < 0.01 and **** p < 0.0001. Scale bars, 100 μm (F and M) , 50 μm (I) , 20 μm ( H and magnified view of I ). The data underlying the graphs shown in the figure can be found in

Article Snippet: For the analysis of cell growth curves, after the addition of 0.1 μM S100B recombinant protein, the cell culture plate was placed in a fully automated live cell dynamic imaging system (Incucyte SX5, Sartorius), and the cell count was recorded every hour for a continuous period of 72 h. Seahorse extracellular flux analysis for glycolytic stress test.

Techniques: Activation Assay, RNA Sequencing, Derivative Assay, Expressing, Control, Staining, Recombinant, Two Tailed Test

Journal: PLOS Biology

Article Title: The suprachiasmatic nucleus regulates brown fat thermogenesis in male mice through an adrenergic receptor ADRB3-S100B signaling pathway

doi: 10.1371/journal.pbio.3003534

Figure Lengend Snippet: (A) S100b expression levels under various nutritional states (fasting, HFD, and TRF) and environmental temperatures (4°C, 21°C, and 30°C). n = 4 per group. (B) S100b and Ucp1 expression levels in indicated temperatures. n = 4 per group. (C) Serum S100B levels were measured by ELISA in sham and SCNx mice under Ad-STE and TRF-STE at ZT4 and ZT16. n = 4 per group. (D and E) Serum biochemical assays showing levels of TG ( D ) and NEFA ( E ) in sham and SCNx mice under Ad-STE ( n = 6 per group). (F) Representative immunohistochemistry images of S100B protein in interscapular BAT from sham and SCNx mice under Ad-STE and TRF-STE conditions. Scale bars, 50μm. (G and H) Western blot analysis ( G ) and densitometric quantification ( H ) of S100B, p21, and CCND1 protein levels in interscapular BAT from sham and SCNx mice at ZT4 and ZT16. n = 4 per group. Red arrows indicate quantified bands. (I) Relative mRNA levels of indicated genes in interscapular BAT from sham and SCNx BAT following ADRB3 antagonist with SR59230A. n = 6 per group. SR: SR59230A. (J and K) Structural prediction of the human ADRB3-S100B complex using AlphaFold2/ColabFold. The human ADRB3/S100B complex model ( J ) was predicted by ColabFold and colored in blue, cyan, yellow, and orange according to different prediction confidence (pLDDT, predicted local distance difference test). Surface electrostatic representation showing ADRB3 (deep teal) and S100B (orange) with positive and negative charges indicated in blue and red, respectively (K) . ( L ) coIP of ADRB3 and S100B in HEK293T cells. (M and N) Western blot ( M ) and densitometry analysis ( N ) of ADRB3 protein levels in interscapular BAT following S100b knockdown or overexpression in vivo. n = 4 per group. Red arrows indicate quantified bands. (O and P) ECAR analysis ( O ) and quantification of basal glycolysis and glycolytic capacity ( P ) in primary preadipocytes treated with S100B and/or β3-agonist CL-316243. Glucose, oligomycin, and 2-DG were sequentially injected. Data are presented as mean ± SEM. Control, CL, 0.1 µM S100B and 0.2 µM S100B: n = 6; 0.1 µM S100B + CL and 0.2 µM S100B+CL: n = 3. (Q) Schematic model illustrating the proposed ADRB3-S100B signaling axis. SCN lesioning enhances SNS activity, thereby promoting ADRB3 signaling and upregulating S100B expression. S100B, in turn, increases ADRB3 sensitivity, establishing a positive feedback loop that sustains thermogenesis and stimulates preadipocyte proliferation. Created in BioRender.com. Unless otherwise indicated, data are presented as mean ± SD. NS: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 by unpaired two-tailed Student t test ( A – E , H , I , N, and P ). The data underlying the graphs shown in the figure can be found in S1 Source Data. Raw blot images can be found in S1 Raw Images.

Article Snippet: For the analysis of cell growth curves, after the addition of 0.1 μM S100B recombinant protein, the cell culture plate was placed in a fully automated live cell dynamic imaging system (Incucyte SX5, Sartorius), and the cell count was recorded every hour for a continuous period of 72 h. Seahorse extracellular flux analysis for glycolytic stress test.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Western Blot, Structural Proteomics, Knockdown, Over Expression, In Vivo, Injection, Control, Activity Assay, Two Tailed Test

Journal: PLOS Biology

Article Title: The suprachiasmatic nucleus regulates brown fat thermogenesis in male mice through an adrenergic receptor ADRB3-S100B signaling pathway

doi: 10.1371/journal.pbio.3003534

Figure Lengend Snippet: (A) Schematic of AAV-mediated S100b knockdown or scramble control delivery into interscapular BAT of SCNx mice. (B) Relative mRNA expression of the indicated genes in interscapular BAT from SCNx mice under TRF-STE with scramble or S100b targeting shRNA. n = 6 per group. (C and D) Western blot analysis of S100B and p21 ( C ) and corresponding densitometry quantification ( D ) in interscapular BAT from SCNx mice under TRF-STE ( n = 4 per group). (E and F) EdU incorporation analysis in interscapular BAT from SCNx mice following S100b knockdown or scramble control. Representative images ( E ) and quantification of EdU-positive nuclei relative to total Hoechst-positive nuclei (F) . (G) Body temperature profiles of SCNx mice with S100b knockdown or scramble control under Ad-STE and TRF-STE (scramble: n = 6, S100b knockdown: n = 5). Data presented as mean ± SEM. Corresponding interscapular BAT and tail temperature data are shown in . (H) Schematic illustrating AAV-mediated overexpression of S100b (AAV-DIO- S100b + AAV-CAG-Cre) or EGFP control in interscapular BAT of WT mice. (I) Relative mRNA levels of indicated genes in interscapular BAT from EGFP or S100b overexpressing mice under TRF-STE ( n = 6 per group). (J and K) Western blot analysis of S100B and p21 proteins ( J ) and corresponding densitometry ( K ) from interscapular BAT of EGFP or S100b -overexpressing mice ( n = 4 per group). (L and M) EdU incorporation analysis in interscapular BAT from EGFP and S100b -overexpressing mice. Representative images ( L ) and quantification of EdU + cells (M) . n = 6 per group. (N) Body temperature responses under Ad-STE and TRF-STE in WT mice with EGFP or S100b overexpression in interscapular BAT. Data presented as mean ± SEM. n = 6 per group. See for interscapular BAT and tail temperature profiles. Unless otherwise indicated, data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. Significance determined by unpaired two-tailed Student t test ( B , D , F , I , K, and M ) or two-way ANOVA with Sidak’S multiple comparisons test (G, N) . Scale bars, 50 μm (E, L) , 20 μm (magnified view of E , L ). Schematic ( A , H ) created in BioRender.com. The data underlying the graphs shown in the figure can be found in S1 Source Data. Raw blot images can be found in S1 Raw Images.

Article Snippet: For the analysis of cell growth curves, after the addition of 0.1 μM S100B recombinant protein, the cell culture plate was placed in a fully automated live cell dynamic imaging system (Incucyte SX5, Sartorius), and the cell count was recorded every hour for a continuous period of 72 h. Seahorse extracellular flux analysis for glycolytic stress test.

Techniques: Knockdown, Control, Expressing, shRNA, Western Blot, Over Expression, Two Tailed Test

Journal: PLOS Biology

Article Title: The suprachiasmatic nucleus regulates brown fat thermogenesis in male mice through an adrenergic receptor ADRB3-S100B signaling pathway

doi: 10.1371/journal.pbio.3003534

Figure Lengend Snippet: (A) LL exposure paradigm is used to disrupt SCN rhythmicity. Arrhythmic mice were selected for subsequent analysis . (B) Relative mRNA expression of indicated genes in interscapular BAT from WT mice under light/dark cycle (LD) or LL during TRF-STE ( n = 4 per group). (C and D) Western blot analysis ( C ) and densitometry ( D ) of S100B and p21 proteins in interscapular BAT from mice under LD or LL conditions ( n = 4 per group). (E and F) EdU staining in interscapular BAT from mice under LD or LL. Representative images ( E ) and quantification of EdU + cells ( F ) normalized to Hoechst staining. (G) Body temperature profiles of WT mice under LD or LL conditions during TRF-STE. Data presented as mean ± SEM. n = 4 per group. See for additional temperature metrics. (H) Working model illustrating the SCN-ADRB3-S100B axis in BAT during TRF conducted at ZT16-ZT20 in a subthermoneutral environment, the SCN regulates BAT thermogenic plasticity via SNS output. SCN lesioning enhances ADRB3 signaling and S100B expression, which together form a positive feedback loop that amplifies β3-adrenergic sensitivity, promotes preadipocyte proliferation, suppresses senescence, and sustains glucose-driven thermogenesis. Unless otherwise indicated, data are presented as mean ± SD. * p < 0.05, ** p < 0.01 and **** p < 0.0001. Significance determined by unpaired two-tailed Student t test ( B , D, and F ) or two-way ANOVA with Sidak’S multiple comparisons test (G) . Scale bars, 50 μm (E) , 20 μm (magnified view of E ). Schematic ( A ) and working model ( H ) created in BioRender.com. The data underlying the graphs shown in the figure can be found in S1 Source Data. Raw blot images can be found in S1 Raw Images.

Article Snippet: For the analysis of cell growth curves, after the addition of 0.1 μM S100B recombinant protein, the cell culture plate was placed in a fully automated live cell dynamic imaging system (Incucyte SX5, Sartorius), and the cell count was recorded every hour for a continuous period of 72 h. Seahorse extracellular flux analysis for glycolytic stress test.

Techniques: Expressing, Western Blot, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Catch-and-Display Immunoassay as an Accessible Platform for Digital Biomarker Detection

doi: 10.64898/2026.01.27.702166

Figure Lengend Snippet: (A) Representative fields of view for the blank sample (0 µg/mL) and S100B positive (0.2 µg/mL) samples. Pronounced colocalization between immunocomplex fluorescence and nanoparticle hotspots is observed only in the presence of S100B. (B) Digital analysis shows dose-dependent response with saturation at 0.75 µg/mL and linear detection from 0.02–0.75 µg/mL (R² = 0.9889). (C) Analog analysis based on total fluorescence enables linear quantification beyond 0.5 µg/mL, complementing digital readout and expanding the dynamic detection range. Data are presented as mean ± SD (N = 6 spots on a single device). Statistical significance was evaluated using one-way ANOVA with Dunnett’s test (****p < 0.0001, ***p = 0.0007).

Article Snippet: The following reagents and materials were used in this study: anti-S100B (TA700560 and TA600560), anti-GFAP antibody pairs (TA700041 and TA600039), and S100B (TP300277) were all purchased from Origene.

Techniques: Fluorescence

Journal: bioRxiv

Article Title: Catch-and-Display Immunoassay as an Accessible Platform for Digital Biomarker Detection

doi: 10.64898/2026.01.27.702166

Figure Lengend Snippet: (A) Representative fields of view of S100B-negative (no S100B input) and S100B-positive samples (0.1 µg/mL). In the presence of S100B, pronounced colocalization between immunocomplex fluorescence and nanoparticle hotspots is observed, indicating successful formation of target-specific immunocomplexes, whereas negligible colocalization is detected in the absence of S100B. (B) Digital Analysis of the TSA-Modified CAD-IA. Dose response analysis demonstrates sensitive and reproducible detection of S100B with a sensitivity down to 0.0002 µg/mL, at least 100-fold higher than CAD-IA not using TSA. Linear regression of the digital signal output shows a strong correlation between colocalization percentage and S100B concentration across the dynamic range of 0.0002–1 µg/mL (R² = 0.9663). Data are presented as mean ± SD (N = 6 spots on a single device). Statistical significance was evaluated using ordinary one-way ANOVA with Dunnett’s multiple comparisons test (****p < 0.0001, ***p = 0.0002).

Article Snippet: The following reagents and materials were used in this study: anti-S100B (TA700560 and TA600560), anti-GFAP antibody pairs (TA700041 and TA600039), and S100B (TP300277) were all purchased from Origene.

Techniques: Fluorescence, Modification, Concentration Assay

Journal: PLOS Biology

Article Title: The suprachiasmatic nucleus regulates brown fat thermogenesis in male mice through an adrenergic receptor ADRB3-S100B signaling pathway

doi: 10.1371/journal.pbio.3003534

Figure Lengend Snippet: (A) Volcano plot showing differentially expressed genes (DEGs) in interscapular BAT from sham and SCNx mice under TRF-STE. DEGs were defined by fold change > 2, p < 0.01. Commonly upregulated genes are highlighted in red and downregulated genes in green at both ZT4 and ZT16. n = 3 per group. (B) IPA of DEGs at ZT4 and ZT16. Pathways with significant enrichment ( p < 0.01) are shown, with activation z-scores indicated for each pathway. (C) DAVID pathway analysis of S100 family-associated DEGs. Enriched KEGG pathways are shown in yellow, and Gene Ontology biological processes (BP) are shown in gray. (D and E) Correlation analysis of RNA-seq-derived S100b expression with cell proliferation markers ( D ) and senescence-related genes ( E ) in sham and SCNx mice. Pearson correlation coefficients and p -values are shown. (F and G) Cell proliferation analysis via EdU incorporation. Representative images showing EdU incorporation in interscapular BAT from sham and SCNx mice at ZT4 and ZT16 under Ad-STE and TRE-STE with 4°C and 30°C as control (F) . Quantification of EdU + nuclei as a percentage of total (Hoechst-stained) nuclei (G) . n = 5 per group. (H–K) Assessment of S100B-induced proliferation in PDGFRα + preadipocytes. PDGFRα-positive stromal vascular fraction (SVF) cells (H) , representative images of EdU-positive cells treated with recombinant S100B (I) , statistical analysis of EdU + nuclei (J) , and cell growth curves of control vs. S100B-treated preadipocytes (K) . (L) Expression of differentiation-related genes during SVF from interscapular BAT induced to differentiate into mature adipocytes. n = 6 per group. (M) Representative β-galactosidase staining images showing senescent cell abundance in interscapular BAT from sham and SCNx mice under TRF-STE. Data are presented as mean ± SD. Statistical significance was determined using unpaired two-tailed Student t test ( G , J, and L ), Pearson correlation analysis ( D and E ), and two-way ANOVA with Sidak’S multiple comparisons test (K) . * p < 0.05, ** p < 0.01 and **** p < 0.0001. Scale bars, 100 μm (F and M) , 50 μm (I) , 20 μm ( H and magnified view of I ). The data underlying the graphs shown in the figure can be found in

Article Snippet: Recombinant Mouse S100B , Targetmol , Cat# TMPJ-00990.

Techniques: Activation Assay, RNA Sequencing, Derivative Assay, Expressing, Control, Staining, Recombinant, Two Tailed Test

Journal: PLOS Biology

Article Title: The suprachiasmatic nucleus regulates brown fat thermogenesis in male mice through an adrenergic receptor ADRB3-S100B signaling pathway

doi: 10.1371/journal.pbio.3003534

Figure Lengend Snippet: (A) S100b expression levels under various nutritional states (fasting, HFD, and TRF) and environmental temperatures (4°C, 21°C, and 30°C). n = 4 per group. (B) S100b and Ucp1 expression levels in indicated temperatures. n = 4 per group. (C) Serum S100B levels were measured by ELISA in sham and SCNx mice under Ad-STE and TRF-STE at ZT4 and ZT16. n = 4 per group. (D and E) Serum biochemical assays showing levels of TG ( D ) and NEFA ( E ) in sham and SCNx mice under Ad-STE ( n = 6 per group). (F) Representative immunohistochemistry images of S100B protein in interscapular BAT from sham and SCNx mice under Ad-STE and TRF-STE conditions. Scale bars, 50μm. (G and H) Western blot analysis ( G ) and densitometric quantification ( H ) of S100B, p21, and CCND1 protein levels in interscapular BAT from sham and SCNx mice at ZT4 and ZT16. n = 4 per group. Red arrows indicate quantified bands. (I) Relative mRNA levels of indicated genes in interscapular BAT from sham and SCNx BAT following ADRB3 antagonist with SR59230A. n = 6 per group. SR: SR59230A. (J and K) Structural prediction of the human ADRB3-S100B complex using AlphaFold2/ColabFold. The human ADRB3/S100B complex model ( J ) was predicted by ColabFold and colored in blue, cyan, yellow, and orange according to different prediction confidence (pLDDT, predicted local distance difference test). Surface electrostatic representation showing ADRB3 (deep teal) and S100B (orange) with positive and negative charges indicated in blue and red, respectively (K) . ( L ) coIP of ADRB3 and S100B in HEK293T cells. (M and N) Western blot ( M ) and densitometry analysis ( N ) of ADRB3 protein levels in interscapular BAT following S100b knockdown or overexpression in vivo. n = 4 per group. Red arrows indicate quantified bands. (O and P) ECAR analysis ( O ) and quantification of basal glycolysis and glycolytic capacity ( P ) in primary preadipocytes treated with S100B and/or β3-agonist CL-316243. Glucose, oligomycin, and 2-DG were sequentially injected. Data are presented as mean ± SEM. Control, CL, 0.1 µM S100B and 0.2 µM S100B: n = 6; 0.1 µM S100B + CL and 0.2 µM S100B+CL: n = 3. (Q) Schematic model illustrating the proposed ADRB3-S100B signaling axis. SCN lesioning enhances SNS activity, thereby promoting ADRB3 signaling and upregulating S100B expression. S100B, in turn, increases ADRB3 sensitivity, establishing a positive feedback loop that sustains thermogenesis and stimulates preadipocyte proliferation. Created in BioRender.com. Unless otherwise indicated, data are presented as mean ± SD. NS: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 by unpaired two-tailed Student t test ( A – E , H , I , N, and P ). The data underlying the graphs shown in the figure can be found in S1 Source Data. Raw blot images can be found in S1 Raw Images.

Article Snippet: Recombinant Mouse S100B , Targetmol , Cat# TMPJ-00990.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Western Blot, Structural Proteomics, Knockdown, Over Expression, In Vivo, Injection, Control, Activity Assay, Two Tailed Test

Journal: PLOS Biology

Article Title: The suprachiasmatic nucleus regulates brown fat thermogenesis in male mice through an adrenergic receptor ADRB3-S100B signaling pathway

doi: 10.1371/journal.pbio.3003534

Figure Lengend Snippet: (A) Schematic of AAV-mediated S100b knockdown or scramble control delivery into interscapular BAT of SCNx mice. (B) Relative mRNA expression of the indicated genes in interscapular BAT from SCNx mice under TRF-STE with scramble or S100b targeting shRNA. n = 6 per group. (C and D) Western blot analysis of S100B and p21 ( C ) and corresponding densitometry quantification ( D ) in interscapular BAT from SCNx mice under TRF-STE ( n = 4 per group). (E and F) EdU incorporation analysis in interscapular BAT from SCNx mice following S100b knockdown or scramble control. Representative images ( E ) and quantification of EdU-positive nuclei relative to total Hoechst-positive nuclei (F) . (G) Body temperature profiles of SCNx mice with S100b knockdown or scramble control under Ad-STE and TRF-STE (scramble: n = 6, S100b knockdown: n = 5). Data presented as mean ± SEM. Corresponding interscapular BAT and tail temperature data are shown in . (H) Schematic illustrating AAV-mediated overexpression of S100b (AAV-DIO- S100b + AAV-CAG-Cre) or EGFP control in interscapular BAT of WT mice. (I) Relative mRNA levels of indicated genes in interscapular BAT from EGFP or S100b overexpressing mice under TRF-STE ( n = 6 per group). (J and K) Western blot analysis of S100B and p21 proteins ( J ) and corresponding densitometry ( K ) from interscapular BAT of EGFP or S100b -overexpressing mice ( n = 4 per group). (L and M) EdU incorporation analysis in interscapular BAT from EGFP and S100b -overexpressing mice. Representative images ( L ) and quantification of EdU + cells (M) . n = 6 per group. (N) Body temperature responses under Ad-STE and TRF-STE in WT mice with EGFP or S100b overexpression in interscapular BAT. Data presented as mean ± SEM. n = 6 per group. See for interscapular BAT and tail temperature profiles. Unless otherwise indicated, data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. Significance determined by unpaired two-tailed Student t test ( B , D , F , I , K, and M ) or two-way ANOVA with Sidak’S multiple comparisons test (G, N) . Scale bars, 50 μm (E, L) , 20 μm (magnified view of E , L ). Schematic ( A , H ) created in BioRender.com. The data underlying the graphs shown in the figure can be found in S1 Source Data. Raw blot images can be found in S1 Raw Images.

Article Snippet: Recombinant Mouse S100B , Targetmol , Cat# TMPJ-00990.

Techniques: Knockdown, Control, Expressing, shRNA, Western Blot, Over Expression, Two Tailed Test

Journal: PLOS Biology

Article Title: The suprachiasmatic nucleus regulates brown fat thermogenesis in male mice through an adrenergic receptor ADRB3-S100B signaling pathway

doi: 10.1371/journal.pbio.3003534

Figure Lengend Snippet: (A) LL exposure paradigm is used to disrupt SCN rhythmicity. Arrhythmic mice were selected for subsequent analysis . (B) Relative mRNA expression of indicated genes in interscapular BAT from WT mice under light/dark cycle (LD) or LL during TRF-STE ( n = 4 per group). (C and D) Western blot analysis ( C ) and densitometry ( D ) of S100B and p21 proteins in interscapular BAT from mice under LD or LL conditions ( n = 4 per group). (E and F) EdU staining in interscapular BAT from mice under LD or LL. Representative images ( E ) and quantification of EdU + cells ( F ) normalized to Hoechst staining. (G) Body temperature profiles of WT mice under LD or LL conditions during TRF-STE. Data presented as mean ± SEM. n = 4 per group. See for additional temperature metrics. (H) Working model illustrating the SCN-ADRB3-S100B axis in BAT during TRF conducted at ZT16-ZT20 in a subthermoneutral environment, the SCN regulates BAT thermogenic plasticity via SNS output. SCN lesioning enhances ADRB3 signaling and S100B expression, which together form a positive feedback loop that amplifies β3-adrenergic sensitivity, promotes preadipocyte proliferation, suppresses senescence, and sustains glucose-driven thermogenesis. Unless otherwise indicated, data are presented as mean ± SD. * p < 0.05, ** p < 0.01 and **** p < 0.0001. Significance determined by unpaired two-tailed Student t test ( B , D, and F ) or two-way ANOVA with Sidak’S multiple comparisons test (G) . Scale bars, 50 μm (E) , 20 μm (magnified view of E ). Schematic ( A ) and working model ( H ) created in BioRender.com. The data underlying the graphs shown in the figure can be found in S1 Source Data. Raw blot images can be found in S1 Raw Images.

Article Snippet: Recombinant Mouse S100B , Targetmol , Cat# TMPJ-00990.

Techniques: Expressing, Western Blot, Staining, Two Tailed Test

Journal: The Journal of Physiology

Article Title: Enteric glial S100B controls rhythmic colonic functions by regulating excitability and specificity in gut motor neurocircuits

doi: 10.1113/JP289410

Figure Lengend Snippet: A , representative images of a myenteric ganglion from the mouse colon labelled with antibodies against the glial markers GFAP (green) and S100B (magenta). Note extensive co‐labelling between GFAP and S100B in the merged image (right panel) showing that cells containing S100B are myenteric glia. Inset highlights one area of this ganglion at higher magnification. B , a representative ganglion co‐labelled with antibodies against S100B (magenta) and the neuronal marker peripherin (green) showing that myenteric neurons do not express S100B. C , total S100B levels in longitudinal muscle‐myenteric plexus (LMMP) tissue samples from the proximal colon and their respective supernatants (medium). Treatment with the S100B synthesis inhibitor arundic acid (50 µM, 1 h) reduced S100B levels in both tissue ( D ) and supernatant ( E ) ( P = 0.0379 and 0.0337, respectively), without affecting cellular markers or colonic cytoarchitecture ( F ). N = 3 mice. Data were analyzed using an unpaired Student's t test and plotted as the mean ± SD. A – F , scale bar = 50 µm. G , representative images of brightfield, neuronal marker PGP9.5 (cyan) and glial marker S100B (magenta) in healthy human colon cross‐sections. Note the colonic layers and the position of myenteric ganglia between circular and longitudinal smooth muscle layers. H , Zoomed images of a myenteric ganglion labeled with markers of neurons and glia showing that S100B labelling is confined to enteric glia as observed in the mouse ENS. G , scale bar = 50 µm. H , scale bar = 100 µm. Muc = mucosa, Subm = submucosa.

Article Snippet: ChAT CreGCaMP5g‐tdTom wholemounts underwent the following sequential treatments: spontaneous recording in Krebs buffer, incubation with arundic acid (50 μ m , 20 min), washout (15 min) and incubation with recombinant S100B protein (50 μg mL −1 ; Novus Bio, Centennial, CO, USA) to verify temporal responses from the same ganglia.

Techniques: Marker, Labeling

Journal: The Journal of Physiology

Article Title: Enteric glial S100B controls rhythmic colonic functions by regulating excitability and specificity in gut motor neurocircuits

doi: 10.1113/JP289410

Figure Lengend Snippet: A , schematic representation showing mechanisms by which arundic acid (50 µ m ) inhibits S100B release in circular muscle‐myenteric plexus whole‐mount preparations. B , representative images of spontaneous activity over time in ganglia from Wnt1 Cre2GCaMP5g‐tdTom mice under control conditions (Krebs buffer) and after arundic acid treatment. Purple arrows indicate enteric glia and white arrows indicate enteric neurons. Effects of arundic acid on the percentages of spontaneously active glia ( C ) and neurons ( G ) and response amplitudes ( D and H ) when data were pooled from both sexes. Stratifying responses by sex shows effects of arundic acid on male and female glial ( P < 0.001, and P < 0.0001, respectively) ( E and F ) and neuronal response amplitudes ( P = 0.128 and 0.0014, respectively) ( I and J ). N = 10 mice; n = 720–1000 neurons and 600–650 glial cells. Data were analyzed using Student's t test, followed by the Mann–Whitney post hoc test. Scale bar = 50 µm. AA = arundic acid.

Article Snippet: ChAT CreGCaMP5g‐tdTom wholemounts underwent the following sequential treatments: spontaneous recording in Krebs buffer, incubation with arundic acid (50 μ m , 20 min), washout (15 min) and incubation with recombinant S100B protein (50 μg mL −1 ; Novus Bio, Centennial, CO, USA) to verify temporal responses from the same ganglia.

Techniques: Activity Assay, Control, MANN-WHITNEY

Journal: The Journal of Physiology

Article Title: Enteric glial S100B controls rhythmic colonic functions by regulating excitability and specificity in gut motor neurocircuits

doi: 10.1113/JP289410

Figure Lengend Snippet: A , schematic illustrating two approaches to disrupt extracellular S100B signalling using pentamidine (PTM) that interferes with S100B–Ca 2 ⁺ interactions and subsequent binding to targets such as RAGE, and an anti‐S100B‐neutralizing antibody. B , representative images of activity over time in ganglia from Wnt1 Cre2GCaMP5g‐tdTom transgenic mice under control conditions (Krebs) and in ganglia following incubation with pentamidine or anti‐S100B antibodies. Purple arrows indicate enteric glia and white arrows indicate enteric neurons. Effects of S100B‐targeted interventions on the percentage of spontaneously active glia ( C ) and neurons ( G ) and their response amplitudes ( P = 0.0063 and P < 0.0001; P < 0.0001 and P < 0.0001, respectively) ( D and H ). Sex stratified data showing effects of PTM and anti‐S100B antibodies on male and female glial ( P = 0.0056 and P < 0.0001, respectively) ( E and F ) and neuronal spontaneous response amplitudes ( P < 0.0001; P < 0.001 and P < 0.0001, respectively) ( I and J ). N = 7 mice; n = 400–720 neurons and 400–600 glial cells. Data were analyzed using the Kruskal–Wallis test followed by Dunn's multiple comparisons test. Scale bar = 50 µm. PTM = pentamidine, S100B ab = S100B antibodies.

Article Snippet: ChAT CreGCaMP5g‐tdTom wholemounts underwent the following sequential treatments: spontaneous recording in Krebs buffer, incubation with arundic acid (50 μ m , 20 min), washout (15 min) and incubation with recombinant S100B protein (50 μg mL −1 ; Novus Bio, Centennial, CO, USA) to verify temporal responses from the same ganglia.

Techniques: Binding Assay, Activity Assay, Transgenic Assay, Control, Incubation

Journal: The Journal of Physiology

Article Title: Enteric glial S100B controls rhythmic colonic functions by regulating excitability and specificity in gut motor neurocircuits

doi: 10.1113/JP289410

Figure Lengend Snippet: Summary data showing that the RAGE antagonist FPS‐ZM1 does not affect colonic motor complex amplitudes ( P = 0.8169 and 0.1504) ( A ), integral ( P = 0.3171 and 0 6362) ( B ), frequency ( P = 0.2801) and propagation velocity ( P = 0.6002) in colons from healthy mice ( C ). N = 4–5 mice for organ bath experiments. Data were analyzed using non‐paired Student's t test. D , representative images of immunolabeling for DAPI (blue), peripherin (green), S100B (magenta) and RAGE (greyscale) in myenteric ganglia from the proximal and distal colons of mice showing that RAGE labelling is scarce in both regions during physiological conditions. Scale bar = 100 µm. E – F , effects of anti‐RAGE‐neutralizing antibodies on spontaneous neuronal activity in myenteric ganglia from Wnt1 Cre2GCaMP5g‐tdTom mice ( P = 0.0566). N = 2 mice; n = ∼300–400 neurons. Data were analyzed using Student's t test, followed by the Mann–Whitney post hoc test. Scale bar in ( E ) = 50 µm. RAGE ab = RAGE antibodies.

Article Snippet: ChAT CreGCaMP5g‐tdTom wholemounts underwent the following sequential treatments: spontaneous recording in Krebs buffer, incubation with arundic acid (50 μ m , 20 min), washout (15 min) and incubation with recombinant S100B protein (50 μg mL −1 ; Novus Bio, Centennial, CO, USA) to verify temporal responses from the same ganglia.

Techniques: Immunolabeling, Activity Assay, MANN-WHITNEY

Journal: The Journal of Physiology

Article Title: Enteric glial S100B controls rhythmic colonic functions by regulating excitability and specificity in gut motor neurocircuits

doi: 10.1113/JP289410

Figure Lengend Snippet: A , representative images of tdTomato (magenta, left), GCaMP5g (green, middle), and spontaneous activity (temporally colour‐coded image, right) in a myenteric ganglion from a Chat CreGCaMP5g‐tdTom mouse. Inset (left) shows a schematic of the Chat CreGCaMP5g‐tdTom transgenic line, which expresses the genetically encoded calcium indicator GCaMP5g specifically in ChAT(+) neurons. B – E , representative maximum intensity projections showing spontaneous neuronal activity over time under control conditions (Krebs) ( B ), following incubation with arundic acid ( C ), post‐arundic acid washout ( D ) and addition of recombinant S100B (rS100B) ( E ). Representative traces of neuron activity are shown on the right. F and G , summary data showing quantification of effects on neuronal response amplitudes during arundic acid incubation ( F ) ( P < 0.0001) and post‐AA ( P < 0.0001) and neuronal firing frequency during and post‐AA ( G ) ( P < 0.0001 and P < 0.001, respectively). N = 360–390 ChAT(+) neurons for Krebs, arundic acid and post‐arundic acid; N = 100 neurons for rS100B. Data were analyzed using the Kruskal–Wallis test followed by Dunn's multiple comparisons test. Scale bar = 50 µm. AA = arundic acid, rS100B = recombinant S100B protein.

Article Snippet: ChAT CreGCaMP5g‐tdTom wholemounts underwent the following sequential treatments: spontaneous recording in Krebs buffer, incubation with arundic acid (50 μ m , 20 min), washout (15 min) and incubation with recombinant S100B protein (50 μg mL −1 ; Novus Bio, Centennial, CO, USA) to verify temporal responses from the same ganglia.

Techniques: Activity Assay, Transgenic Assay, Control, Incubation, Recombinant

Journal: The Journal of Physiology

Article Title: Enteric glial S100B controls rhythmic colonic functions by regulating excitability and specificity in gut motor neurocircuits

doi: 10.1113/JP289410

Figure Lengend Snippet: A , temporally colour‐coded images showing spontaneous activity among cholinergic neurons in a myenteric ganglion from a Chat CreGCaMP5g‐tdTom mouse under control conditions (Krebs) and following incubation in arundic acid. Note that inhibiting S100B release reduces the diversity of spontaneous oscillations in ChAT(+) neurons over time. This effect is illustrated by dendrograms ( B ) that show clusters of ChAT(+) neurons grouped based on pairwise correlations. Summary data ( C ) reveal treating samples with arundic acid produces fewer distinct clusters among ChAT(+) neurons ( P = 0.00720). D , Z‐stacked average intensity images of activity over time show pairs of neurons becoming highly active and correlated following S100B inhibition. Zoomed regions ( Dd ′ and Dd ′′ ) highlight similar response patterns between neuron pairs. E , representative heatmaps displaying the strength and extent of pairwise correlations among cholinergic neurons show that arundic acid increases correlated (green) and highly correlated (yellow) neuron pairs. F and G , summary data showing effects of arundic acid on normalized pairwise correlations ( P = 0.0181) and Earth Mover's Distance (EMD) indices ( P = 0.131) ( F ) and their combination (global synchronization) ( P = 0.00780) ( G ). H , representative waterfall plots showing effects of arundic acid on temporal activity patterns of clustered cholinergic neurons. Ganglia: N = 12 or 13 from four animals; n cells = ∼500. Data were analyzed using an unpaired Student's t test. Scale bars = 50 µm ( A and D ); 25 µm ( d′ and d′′ ). AA = arundic acid.

Article Snippet: ChAT CreGCaMP5g‐tdTom wholemounts underwent the following sequential treatments: spontaneous recording in Krebs buffer, incubation with arundic acid (50 μ m , 20 min), washout (15 min) and incubation with recombinant S100B protein (50 μg mL −1 ; Novus Bio, Centennial, CO, USA) to verify temporal responses from the same ganglia.

Techniques: Activity Assay, Control, Incubation, Inhibition