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: S100B and VEGFA levels in the supernatant were measured using S100B (Elabscience, E-EL-H1297) and VEGFA (Elabscience, E-EL-H0111) ELISA kits.

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: S100B and VEGFA levels in the supernatant were measured using S100B (Elabscience, E-EL-H1297) and VEGFA (Elabscience, E-EL-H0111) ELISA kits.

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: S100B and VEGFA levels in the supernatant were measured using S100B (Elabscience, E-EL-H1297) and VEGFA (Elabscience, E-EL-H0111) ELISA kits.

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: S100B and VEGFA levels in the supernatant were measured using S100B (Elabscience, E-EL-H1297) and VEGFA (Elabscience, E-EL-H0111) ELISA kits.

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: S100B and VEGFA levels in the supernatant were measured using S100B (Elabscience, E-EL-H1297) and VEGFA (Elabscience, E-EL-H0111) ELISA kits.

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: S100B and VEGFA levels in the supernatant were measured using S100B (Elabscience, E-EL-H1297) and VEGFA (Elabscience, E-EL-H0111) ELISA kits.

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: S100B and VEGFA levels in the supernatant were measured using S100B (Elabscience, E-EL-H1297) and VEGFA (Elabscience, E-EL-H0111) ELISA kits.

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

Journal: Scientific reports

Article Title: A nonadjuvanted HLA-restricted peptide vaccine induced both T and B cell immunity against SARS-CoV-2 spike protein.

doi: 10.1038/s41598-024-71663-1

Figure Lengend Snippet: Fig. 4. In vivo immune responses of injected UC peptides and complement activity of immunized serum. (a) BALB/c mice were intramuscularly administered 3 doses of 50 µg of UC-100 (n = 3) or 400 µg of UC-152 (n = 4) 14 days apart. (b–d) The serum of UC-100 mice was collected pre-(Day-1) and posttreatment (Day41) and tested in duplicate and represented as the mean for the levels of (b) total IgG; (c) specific IgG against UC-100 peptide; (d) specific IgG against SARS-CoV-2 spike trimer protein. (e–g) The serum of UC-152 mice was collected pre-(Day-1) and posttreatment (Day41) and tested in duplicate and represented as the mean for the levels of (e) total IgG; (f) specific IgG against UC-152 peptide; (g) specific IgG against SARS-CoV-2 spike trimer protein. (h,i) Antibody-mediated complement activity was tested in duplicate and is represented as the mean for pre- and postserum of mice vaccinated with UC-100 and UC-152 peptides, respectively. The complement activity was expressed as the IC50 of cytotoxicity of the (h) UC-100-vaccinated group and (i) UC-152-vaccinated group. Significant differences are indicated by asterisks (unpaired one-tailed Student’s t test; *p < 0.05, **p < 0.01, ***p < 0.001).

Article Snippet: A final concentration of 10% young rabbit complement (CL3441-S50-R, Cedarlane, Ontario, Canada) was added to each well and incubated for an additional 30 min at 37 °C and 5% CO2.

Techniques: In Vivo, Injection, Activity Assay, One-tailed Test

Journal: Scientific reports

Article Title: A nonadjuvanted HLA-restricted peptide vaccine induced both T and B cell immunity against SARS-CoV-2 spike protein.

doi: 10.1038/s41598-024-71663-1

Figure Lengend Snippet: Fig. 5. In vivo immune responses of oral UC peptides and complement activity of immunized serum. (a) BALB/c mice (n = 5) were orally administered 3 doses of 200 µg UC-100 14 days apart. (b–d) The serum of UC-100 mice was collected pre-(Day-1) and posttreatment (Day 41) and tested in duplicate and represented as the mean for the levels of (b) total IgG; (c) specific IgG against UC-100 peptide; (d) specific IgG against SARS-CoV-2 spike trimer protein; (e) The antibody-mediated complement activity was tested in duplicate and represented as the mean for pre- and postserum of mice vaccinated with UC-100. The complement activity was expressed as the IC50 of cytotoxicity. (f,g) The serum of UC-100 mice was collected pre-(Day-1) and posttreatment (Day 41) and tested in duplicate and represented as the mean for the levels of (f) total IgA and (g) specific IgA against UC-100 peptide. Significant differences are indicated by asterisks (unpaired one-tailed Student’s t test; *p < 0.05, **p < 0.01, ***p < 0.001).

Article Snippet: A final concentration of 10% young rabbit complement (CL3441-S50-R, Cedarlane, Ontario, Canada) was added to each well and incubated for an additional 30 min at 37 °C and 5% CO2.

Techniques: In Vivo, Activity Assay, One-tailed Test

Journal: Scientific reports

Article Title: A nonadjuvanted HLA-restricted peptide vaccine induced both T and B cell immunity against SARS-CoV-2 spike protein.

doi: 10.1038/s41598-024-71663-1

Figure Lengend Snippet: Fig. 6. Summary of the SARS-CoV-2 HLA-restricted peptide T-cell vaccine. The possible mechanistic immune response to HLA-restricted peptides has been elucidated. The 9-mer UC peptides can be primed onto both HLA class I and II molecules, and the immune response is triggered for both humoral and cellular immunity. Through HLA class I molecule binding, CTLs can be activated, and an increase in IFN-γ cytokines and specific lytic activity have been observed. Through HLA class II molecule binding, B-cells are activated, and specific antibodies are produced in accordance with complement cytotoxicity activity.

Article Snippet: A final concentration of 10% young rabbit complement (CL3441-S50-R, Cedarlane, Ontario, Canada) was added to each well and incubated for an additional 30 min at 37 °C and 5% CO2.

Techniques: Binding Assay, Activity Assay, Produced

Journal: Oncotarget

Article Title: Pancreatic cancer-derived exosomes promote tumor metastasis and liver pre-metastatic niche formation

doi: 10.18632/oncotarget.18831

Figure Lengend Snippet: Confocal microscopy (lung, liver, spleen, brain) of PKH67-labeled Panc02 EXO and Panc02-H7 EXO tissue distribution (green) 24 hpi. (A) PKH-67-labeled liposomes served as controls (scale bar=100 μm). Histogram shows exosome tissue distribution quantification (n=5/group). CD45, p-Stat3, and CD11b IF staining in liver sections from controls (left) and mice treated with Panc02 EXOs(middle) or Panc02-H7 EXOs (right) for 12 d without tumor challenge. (B) Histogram shows infiltrating CD45 + cell quantification. FN and α-SMA IF staining in liver sections from controls (top) and mice treated with Panc02 EXOs(middle) or Panc02-H7 EXOs (bottom) for 12 d without tumor challenge. (C) Histogram shows infiltrating α-SMA + hStCs and FN expression quantification(400× magnification; n=5/group). Western blotting analysis showed upregulated S100A8 and S100A9 in livers treated with Panc02-H7-derived exosomes. Histogram shows expression of the three proteins in three groupsas determined by densitometric analysis (n=3/group). (D) Pancreatic cancer-derived exosomes induce MDSC accumulation in peripheral blood. (E) Representative flow cytometric plots (left) and quantification (right) of CD11b + GR1 + MDSCs (n=5/group). *P<0.05, **P<0.01,***P<0.001.

Article Snippet: Primary antibodies against fibronectin (1:100),α-SMA (1:50), S100A8 (1:50), S100A9 (1:50), and F4/80 (1:50) were purchased from Proteintech (Wuhan, China).

Techniques: Confocal Microscopy, Labeling, Liposomes, Staining, Expressing, Western Blot, Derivative Assay

Journal: Oncotarget

Article Title: Pancreatic cancer-derived exosomes promote tumor metastasis and liver pre-metastatic niche formation

doi: 10.18632/oncotarget.18831

Figure Lengend Snippet: IHC analysis and histopathological examination of macrophages (F4/80), hStCs (α-SMA), and neutrophils in liver metastatic niches of naïve mice and mice treated with PBS, Panc02 EXOs, or Panc02-H7 EXOs at 30d post-SOI (arrow shows neutrophils in liver) (A) . Representative histogram shows quantification of F4/80 + macrophages, α-SMA + hStCs, and neutrophils (B) . Identification of FN, S100A8, and S100A9 as inflammatory mediators, and collagen deposition in the liver metastatic niche (C) . Representative histogram shows FN and MTS quantification (D) . Representative histogram shows S100A8 and S100A9 quantification (E) . n=6/group.**P<0.01,***P<0.001.10 fields assessed per sample. FOV, field of view.

Article Snippet: Primary antibodies against fibronectin (1:100),α-SMA (1:50), S100A8 (1:50), S100A9 (1:50), and F4/80 (1:50) were purchased from Proteintech (Wuhan, China).

Techniques:

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: S100A9 promotes resistance to anti-PD-1 immunotherapy in hepatocellular carcinoma by degrading PARP1 and activating the STAT3/PD-L1 pathway

doi: 10.1007/s13402-025-01087-0

Figure Lengend Snippet: S100A9 is highly expressed in anti-PD-1 non-responders and is associated with poor prognosis in HCC. ( A ) Schematic overview of the sample collection and RNA-seq analysis workflow. ( B ) Volcano plots showing differentially expressed genes in tumor vs. adjacent non-tumor tissues (x-axis) and in non-responders vs. responders to anti-PD-1 therapy (y-axis). ( C ) Venn diagram illustrating genes that are upregulated in tumors and non-responders, and also associated with both overall survival (OS) and relapse-free survival (RFS) in the in-house cohort. ( D ) Representative immunohistochemistry (IHC) images showing S100A9 expression in tumor tissues from responders and non-responders. ( E – J ) High S100A9 expression is associated with worse overall survival in GSE202069 ( E ), GSE14520 ( G ), TCGA ( I ), and CHCC ( J ), and with worse relapse-free survival in GSE202069 ( F ) and GSE14520 ( H )

Article Snippet: The S100A9 levels in the culture supernatant were determined using the S100A9 ELISA kit provided by Wuhan Proteintech Biotechnology Co., Ltd. To ensure the accuracy and reliability of the data, experiments were conducted strictly according to the manufacturer’s instructions.

Techniques: RNA Sequencing, Immunohistochemistry, Expressing

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: S100A9 promotes resistance to anti-PD-1 immunotherapy in hepatocellular carcinoma by degrading PARP1 and activating the STAT3/PD-L1 pathway

doi: 10.1007/s13402-025-01087-0

Figure Lengend Snippet: S100A9 inhibits the efficacy of anti-PD-1 therapy in HCC mouse models. ( A ) Schematic illustration of the establishment of subcutaneous Hepa1-6 tumor models and treatment regimen. ( B ) Representative images of subcutaneous tumors from each treatment group. ( C ) Tumor growth curves of Hepa1-6 tumors in C57BL/6 mice treated with either IgG or anti-PD-1 antibody, with or without S100a9 overexpression. ( D ) Schematic diagram depicting the generation of the hydrodynamic tail vein injection (HTVi) model and treatment strategy. ( E ) Representative gross liver tumor images from each group. ( F ) Liver-to-body weight ratios following IgG or anti-PD-1 treatment. ( G ) Quantification of tumor numbers per group post-treatment. ( H ) Representative IHC staining of mouse tumor sections for H&E, S100a9, CD4, and CD8. ( I ) Quantification of CD4⁺ and CD8⁺ T cell infiltration in tumor tissues from each group

Article Snippet: The S100A9 levels in the culture supernatant were determined using the S100A9 ELISA kit provided by Wuhan Proteintech Biotechnology Co., Ltd. To ensure the accuracy and reliability of the data, experiments were conducted strictly according to the manufacturer’s instructions.

Techniques: Over Expression, Injection, Immunohistochemistry

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: S100A9 promotes resistance to anti-PD-1 immunotherapy in hepatocellular carcinoma by degrading PARP1 and activating the STAT3/PD-L1 pathway

doi: 10.1007/s13402-025-01087-0

Figure Lengend Snippet: S100A9 enhances PD–L1 transcription via activation of the STAT3 signaling pathway. ( A ) Gene Set Enrichment Analysis (GSEA) of S100A9- and PD–L1-associated HALLMARK pathways based on the TCGA LIHC cohort. ( B – C ) GSEA indicates a positive correlation between IL-6/JAK/STAT3 signaling and the expression of S100A9 ( B ) and PD–L1 ( C ). ( D ) Western blot analysis of MHCC-97 H cells showing that S100A9 overexpression increases STAT3 phosphorylation at Tyr705 and upregulates PD–L1 protein levels. ( E ) Quantitative PCR analysis confirms that S100A9 overexpression enhances PD–L1 mRNA expression. ( F ) Representative IHC images of HCC clinical and Hepa1-6 mouse tumor samples demonstrating expression patterns of S100A9, phosphorylated STAT3 (Tyr705), and PD–L1

Article Snippet: The S100A9 levels in the culture supernatant were determined using the S100A9 ELISA kit provided by Wuhan Proteintech Biotechnology Co., Ltd. To ensure the accuracy and reliability of the data, experiments were conducted strictly according to the manufacturer’s instructions.

Techniques: Activation Assay, Expressing, Western Blot, Over Expression, Phospho-proteomics, Real-time Polymerase Chain Reaction

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: S100A9 promotes resistance to anti-PD-1 immunotherapy in hepatocellular carcinoma by degrading PARP1 and activating the STAT3/PD-L1 pathway

doi: 10.1007/s13402-025-01087-0

Figure Lengend Snippet: S100A9 interacts with PARP1 and promotes its degradation via the ubiquitin-proteasome pathway. ( A ) Schematic flowchart illustrating the process of immunoprecipitation followed by mass spectrometry (IP–MS). ( B ) Coomassie brilliant blue staining showing successful immunoprecipitation of S100A9 in 293 T cells. ( C ) LC–MS analysis identifying candidate proteins interacting with S100A9. ( D ) Co-immunoprecipitation and Western blotting in MHCC-97 H cells confirm the interaction between S100A9 and PARP1. ( E ) Immunofluorescence staining demonstrates co-localization of S100A9 and PARP1 in Huh7 cells. ( F ) Western blot showing that overexpression of S100A9 downregulates PARP1 protein levels in MHCC-97 H cells. ( G ) Knockdown of S100A9 in Huh7 cells increases PARP1 expression and decreases STAT3 phosphorylation (Tyr705) and PD–L1 expression, as shown by Western blot. ( H ) Schematic representation of PARP1 functional domains. ( I ) Co-immunoprecipitation assays identifying the BRCT domain as the interacting region with S100A9. ( J ) Cycloheximide (CHX) chase assay showing the degradation kinetics of PARP1 and PD–L1 upon S100A9 overexpression. ( K ) Quantification of CHX assays showing normalized PARP1 and PD–L1 protein levels over time. ( L ) Western blot analysis showing that MG132 treatment reverses the reduction of PARP1 induced by S100A9 overexpression in MHCC-97 H cells. ( M – N ) Ubiquitination assays in 293 T cells co-transfected with HA–Ub and either Flag-S100A9 (M) or S100A9 siRNA (N), with or without MG132 treatment. PARP1 was immunoprecipitated, and ubiquitination was assessed using an anti-Ub antibody

Article Snippet: The S100A9 levels in the culture supernatant were determined using the S100A9 ELISA kit provided by Wuhan Proteintech Biotechnology Co., Ltd. To ensure the accuracy and reliability of the data, experiments were conducted strictly according to the manufacturer’s instructions.

Techniques: Ubiquitin Proteomics, Immunoprecipitation, Mass Spectrometry, Protein-Protein interactions, Staining, Liquid Chromatography with Mass Spectroscopy, Western Blot, Immunofluorescence, Over Expression, Knockdown, Expressing, Phospho-proteomics, Functional Assay, Transfection

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: S100A9 promotes resistance to anti-PD-1 immunotherapy in hepatocellular carcinoma by degrading PARP1 and activating the STAT3/PD-L1 pathway

doi: 10.1007/s13402-025-01087-0

Figure Lengend Snippet: Tasquinimod, an S100A9 inhibitor, enhances anti-PD-1 immunotherapy efficacy in a mouse model of HCC. ( A ) Western blot analysis showing the effects of increasing concentrations of the S100A9 inhibitor Tasquinimod on the protein levels of S100A9, PARP1, total STAT3, phosphorylated STAT3 (Tyr705), and PD–L1 in Huh7 cells. ( B ) Schematic diagram illustrating the drug treatment protocol in C57BL/6 mice, including administration of Tasquinimod and/or anti-PD-1 antibody. ( C ) Representative images of subcutaneous Hepa1-6 tumors from each treatment group. ( D ) Tumor growth curves of subcutaneous Hepa1-6 tumors in C57BL/6 mice treated with Tasquinimod, anti-PD-1 antibody, or the combination. ( E ) Representative immunohistochemistry (IHC) staining of mouse tumor samples, including H&E, Ki67, CD4, and CD8. ( F ) Quantification of CD4⁺ and CD8⁺ T cell infiltration in tumor tissues based on IHC analysis. ( G ) Flow cytometry analysis showing CD4⁺ and CD8⁺ T cell infiltration in the indicated treatment groups

Article Snippet: The S100A9 levels in the culture supernatant were determined using the S100A9 ELISA kit provided by Wuhan Proteintech Biotechnology Co., Ltd. To ensure the accuracy and reliability of the data, experiments were conducted strictly according to the manufacturer’s instructions.

Techniques: Western Blot, Immunohistochemistry, Flow Cytometry

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: S100A9 promotes resistance to anti-PD-1 immunotherapy in hepatocellular carcinoma by degrading PARP1 and activating the STAT3/PD-L1 pathway

doi: 10.1007/s13402-025-01087-0

Figure Lengend Snippet: Schematic diagram illustrating the mechanism by which S100A9 promotes resistance to anti-PD-1 immunotherapy in HCC. S100A9 directly interacts with PARP1 and promotes its degradation through the ubiquitin-proteasome pathway, resulting in enhanced phosphorylation of STAT3 at Tyr705 and subsequent transcriptional upregulation of PD–L1. This signaling cascade contributes to immune evasion and resistance to anti-PD-1 therapy. Notably, pharmacological inhibition of S100A9 with Tasquinimod significantly enhances the efficacy of anti-PD-1 treatment in HCC. (Figure created using BioRender.com)

Article Snippet: The S100A9 levels in the culture supernatant were determined using the S100A9 ELISA kit provided by Wuhan Proteintech Biotechnology Co., Ltd. To ensure the accuracy and reliability of the data, experiments were conducted strictly according to the manufacturer’s instructions.

Techniques: Ubiquitin Proteomics, Phospho-proteomics, Inhibition

Journal: Oncology reports

Article Title: Ca2+-binding protein S100A11: a novel diagnostic marker for breast carcinoma.

doi: 10.3892/or_00000764

Figure Lengend Snippet: Figure 2. Comparison of the expression of S100A11 between IDCA (B) and corresponding adjacent normal tissue (A). The arrow points to the location of the spots representing S100A11 and proteins that were up-regulated (>3-fold) in all of the comparison groups under stringent selection conditions. Visualization is by silver staining. The letter u before the number means ‘up-regulated’ and the number is the corresponding marker in each group of differential comparisons between cancer and adjacent normal tissue.

Article Snippet: After blocking with TBS containing 5% milk powder and 0.1% Tween-20 for 1 h at RT, the membranes were incubated with the anti-S100A11 antibody (ProteinTech Group, Inc.) at the concentration ratio of 1:1000 for 1 h at RT followed by treatment with horseradish peroxidase-conjugated secondary antibody for 1 h at RT.

Techniques: Comparison, Expressing, Selection, Silver Staining, Marker

Journal: Oncology reports

Article Title: Ca2+-binding protein S100A11: a novel diagnostic marker for breast carcinoma.

doi: 10.3892/or_00000764

Figure Lengend Snippet: Figure 3. S100A11 protein identified by MALDI-TOF/TOF. (A) A full scan MS-MS spectrum of protein S100A11. (B) Matched peptides are shown in panes (sequence coverage, 21%).

Article Snippet: After blocking with TBS containing 5% milk powder and 0.1% Tween-20 for 1 h at RT, the membranes were incubated with the anti-S100A11 antibody (ProteinTech Group, Inc.) at the concentration ratio of 1:1000 for 1 h at RT followed by treatment with horseradish peroxidase-conjugated secondary antibody for 1 h at RT.

Techniques: Tandem Mass Spectroscopy, Sequencing

Journal: Oncology reports

Article Title: Ca2+-binding protein S100A11: a novel diagnostic marker for breast carcinoma.

doi: 10.3892/or_00000764

Figure Lengend Snippet: Figure 4. Western blot analysis confirmed the expression of S100A11 in breast tumor tissues and adjacent normal tissues. The letter C means ‘group of cancer tissue’ and the letter N means ‘group of normal tissue’. Numbers 1-5 correspond to different subtypes (Table I). (A) Western blot for human tissues. Comparison between breast carcinoma and adjacent normal tissue. (B) Integrated density value (IDV).

Article Snippet: After blocking with TBS containing 5% milk powder and 0.1% Tween-20 for 1 h at RT, the membranes were incubated with the anti-S100A11 antibody (ProteinTech Group, Inc.) at the concentration ratio of 1:1000 for 1 h at RT followed by treatment with horseradish peroxidase-conjugated secondary antibody for 1 h at RT.

Techniques: Western Blot, Expressing, Comparison

Journal: Oncology reports

Article Title: Ca2+-binding protein S100A11: a novel diagnostic marker for breast carcinoma.

doi: 10.3892/or_00000764

Figure Lengend Snippet: Figure 5. Representative images of S100A11 immunostaining in breast tissues. Scale bar, 50 μm. (A) S100A11 positivity in breast tumor. The staining pattern is predominantly cytoplasmic (magnification, x200). (B) S100A11 positivity in corresponding adjacent normal tissue. The staining pattern is predominantly cytoplasmic (magnification, x200). (C) S100A11 positivity in breast adenosis tissue. The staining pattern is predominantly cytoplasmic (magnification, x200).

Article Snippet: After blocking with TBS containing 5% milk powder and 0.1% Tween-20 for 1 h at RT, the membranes were incubated with the anti-S100A11 antibody (ProteinTech Group, Inc.) at the concentration ratio of 1:1000 for 1 h at RT followed by treatment with horseradish peroxidase-conjugated secondary antibody for 1 h at RT.

Techniques: Immunostaining, Staining

Journal: The journal of headache and pain

Article Title: Inhibition of S100A4 decreases neurotoxic astrocyte reactivity and attenuates neuropathic pain via the TLR4/NF-κB pathway in a rat model of spinal nerve ligation.

doi: 10.1186/s10194-025-02045-9

Figure Lengend Snippet: Fig. 1 SNL induces elevated S100A4 expression in the SDH, and niclosamide attenuates NP. Western blot analysis revealed the temporal profile of S100A4 expression in both sham and SNL rats (A). SNL induced elevated S100A4 levels in ipsilateral L4/5 spinal cord segments from Days 1 to 21 after surgery (B). The data are presented as the means ± SEMs. ****p < 0.0001 versus the sham group. Real-time PCR (C) revealed similar trends in S100A4 mRNA expression at 3, 5, 7 and 10 days in SNL rats. The data are presented as the means ± SEMs. ****p < 0.0001 versus the sham group. Immunofluorescence assays revealed the distribution of S100A4 (green) protein in the L4/5 SDH after sham or SNL surgery on POD 10 (D1, D2, and D3; scale bar, 500 μm). The fluorescence in tensity significantly increased at 14 days after SNL and remained high at 21 dpi (D4, D5) (scale bar, 500 μm). Data analysis further confirmed these results, as shown in E. Data are presented as the mean ± SEM. **p < 0.01, ****p < 0.0001, ipsilateral SDH versus sham SDH; ####P < 0.0001, ipsilateral SDH versus contralateral SDH. Double immunofluorescence staining images (F1, F2, and F3) revealed extensive coexpression of S100A4 (green) with NeuN (red) and limited coexpression with GFAP (red) but not with iba-1 (scale bar, 500 μm). More details are shown at higher magnification for F1-1, F2-1 and F3-1 (scale bar, 100 μm). Single intrathecal administration of niclosamide (15–45 µg in 20 µl) in the later phase (POD 7) partially suppressed mechanical hypersensi tivity in the ipsilateral hind paw for up to 5 days after SNL, and the effect was observed 12 h after injection. Moreover, 45 µg of niclosamide had a greater effect than 15 µg of niclosamide (G). The data are presented as the means ± SEMs. ****p < 0.0001 for 15 µg of niclosamide versus DMSO. ####p < 0.0001, 45 µg of niclosamide versus DMSO. ψψψp < 0.001, ψψψψp < 0.0001, 15 µg of niclosamide versus 45 µg of niclosamide. Repeated administration of ni closamide in the early phase (POD 1) partially attenuated the development of mechanical hypersensitivity in SNL rats as well (I), which was maintained until POD 12. The data are presented as the means ± SEMs. ****p < 0.0001 for niclosamide versus DMSO. However, niclosamide treatment did not affect mechanical pain sensitivity in the contralateral hind paw (H, J). As shown in K, a single intrathecal injection of the rat S100A4 peptide (200 ng or 800 ng in 20 µl) rapidly decreased the PWT in the bilateral hind paws of naive rats from 2 h to 7 d. Moreover, 800 ng S100A4 induced more severe hyperalgesia than did 200 ng S100A4. On the other hand, saline did not affect the PWT of the hind paw. The data are presented as the means ± SEMs. ****p < 0.0001, 200 ng of S100A4 versus saline. ####p < 0.0001 800 ng S100A4 versus saline. ψψψψp < 0.0001 200 ng S100A4 versus 800 ng S100A4

Article Snippet: Antibodies against S100A4 (16105-1-AP), C3 (21337- 1-AP), NF-κB p65 (10745-1-AP), and TLR4 (19811-1- AP) were purchased from Proteintech (Hubei, Wuhan, China).

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Immunofluorescence, Fluorescence, Double Immunofluorescence Staining, Injection, Saline

Journal: The journal of headache and pain

Article Title: Inhibition of S100A4 decreases neurotoxic astrocyte reactivity and attenuates neuropathic pain via the TLR4/NF-κB pathway in a rat model of spinal nerve ligation.

doi: 10.1186/s10194-025-02045-9

Figure Lengend Snippet: Fig. 3 Exogenous S100A4 significantly contributes to A1 astrocyte activation in vitro. Western blot analysis revealed that GFAP and C3 protein levels were significantly increased after stimulation of 70% fusion primary astrocyte cultures with different concentrations of rat recombinant S100A4 protein (A), whereas 80 ng/ml S100A4 stimulation induced the greatest increase in GFAP and C3 expression (B). The data are presented as the means ± SEMs. *p < 0.05, **p < 0.01, ****p < 0.0001, normal versus S100A4. ##p < 0.01, ###p < 0.001, ####p < 0.0001 versus 80 ng/ml S100A4. RT‒PCR analysis revealed changes in the mRNA levels of A1-specific astrocyte genes after 80 ng/ml S100A4 stimulation, similar to the effects of the commonly known “cytokine cocktail” containing TNF-α, IL-1α and C1q (C). The data are presented as the means ± SEMs. *p < 0.05, ****p < 0.0001 S100A4 versus normal. #p < 0.05, ###p < 0.001, ####p < 0.0001 TNF-α, IL-1α and C1q versus normal. Moreover, incubation with 80 ng/ml S100A4 resulted in higher OD values than those of normal cells and cells incubated with other concentrations of S100A4 (D). The data are presented as the means ± SEMs. ****p < 0.0001 versus 80 ng/ml S100A4. Im munofluorescence revealed that 70% of the fusion primary astrocytes (GFAP, green; DAPI, blue) incubated with TNF-α, IL-1α and c1q and S100A4 (80 ng/ ml) for 24 h presented stronger GFAP (green) and C3 (red) fluorescence intensities and, accordingly, showed higher GFAP+C3+ fluorescence intensities (E) (scale bar, 100 μm). Quantitative analysis further confirmed these results (F). The data are presented as the means ± SEMs. ****p < 0.0001, normal versus S100A4. ####p < 0.0001, normal versus TNF-α, IL-1α and C1q

Article Snippet: Antibodies against S100A4 (16105-1-AP), C3 (21337- 1-AP), NF-κB p65 (10745-1-AP), and TLR4 (19811-1- AP) were purchased from Proteintech (Hubei, Wuhan, China).

Techniques: Activation Assay, In Vitro, Western Blot, Recombinant, Expressing, Incubation, Fluorescence

Journal: The journal of headache and pain

Article Title: Inhibition of S100A4 decreases neurotoxic astrocyte reactivity and attenuates neuropathic pain via the TLR4/NF-κB pathway in a rat model of spinal nerve ligation.

doi: 10.1186/s10194-025-02045-9

Figure Lengend Snippet: Fig. 4 S100A4 inhibition reduces A1 astrocyte activation following SNL. Western blot analysis revealed significantly reduced levels of GFAP and C3 at 10 and 14 days post-SNL in rats treated with successive niclosamide injections, starting from POD 1, along with decreased S100A4 expression (A and B). The data are presented as the means ± SEMs. ***p < 0.001, ****p < 0.0001, normal versus SNL + DMSO. #p < 0.05, ##p < 0.01, ####p < 0.0001, SNL + DMSO versus SNL + niclosamide. In addition, fluorescence images confirmed that astrocytes were remarkably activated in the SDH after SNL, and the fluorescence in tensity of GFAP (C3) (green) was much greater than that in the sham group (C1) (scale bar, 500 μm). Successive intrathecal injection of niclosamide begin ning on POD1 for 3 days, but not DMSO, suppressed SNL-induced activation of astrocytes (C3 and C4). However, niclosamide had no effect on sham rats (C2) (scale bar, 500 μm). The data summary further confirmed these results, as shown in D. Moreover, niclosamide treatment reduced the fluorescence intensity of GFAP+C3+ (yellow) in SNL rats (E and F). The data are presented as the means ± SEMs. ****p < 0.0001, sham + DMSO group versus SNL + DMSO group. ###p < 0.001, SNL + DMSO versus SNL + niclosamide. ELISA (G) revealed that the administration of niclosamide also decreased the protein levels of proinflammatory cytokines (TNF-α, IL-6, and IL-1β), while RT‒PCR data confirmed that the injection of niclosamide decreased the mRNA levels of A1 astrocyte-specific factors, including CCL2, CXCL1, MMP2, and tPA (H). The data are presented as the means ± SEMs. ****p < 0.0001, sham + DMSO versus SNL + DMSO. ##p < 0.01, ###p < 0.001, ####p < 0.0001 SNL + DMSO versus SNL + niclosamide. Finally, pain behavioural assessment revealed that the ad ministration of recombinant S100A4 protein decreased the PWT of the bilateral hind paws from 2 h to 7 days, whereas fluorocitrate partially reversed S100A4-induced mechanical hyperalgesia, which persisted for up to 7 days (I). The data are presented as the means ± SEMs. ****p < 0.0001, saline versus S100A4. ##p < 0.01, ###p < 0.001, ####p < 0.0001 S100A4 versus S100A4 + fluorocitrate

Article Snippet: Antibodies against S100A4 (16105-1-AP), C3 (21337- 1-AP), NF-κB p65 (10745-1-AP), and TLR4 (19811-1- AP) were purchased from Proteintech (Hubei, Wuhan, China).

Techniques: Inhibition, Activation Assay, Western Blot, Expressing, Fluorescence, Injection, Enzyme-linked Immunosorbent Assay, Recombinant, Saline

Journal: The journal of headache and pain

Article Title: Inhibition of S100A4 decreases neurotoxic astrocyte reactivity and attenuates neuropathic pain via the TLR4/NF-κB pathway in a rat model of spinal nerve ligation.

doi: 10.1186/s10194-025-02045-9

Figure Lengend Snippet: Fig. 5 Exogenous S100A4 induces A1 astrocyte activation via the TLR4/NF-κB signalling pathway in vitro. Western blot analysis revealed that TLR4 and p-NF-κB p65 protein levels were also significantly increased after stimulation with 80 ng/ml rat recombinant S100A4 protein in primary astrocyte cultures, whereas total NF-κB p65 expression was not affected (A, B). The data are presented as the means ± SEMs. **p < 0.01, ****p < 0.0001, normal versus S100A4. Pretreatment with PDTC decreased the S100A4-induced upregulation of GFAP and C3, as well as p-NF-κB p65 expression (C). The selective TLR4 inhibitor Resatorvid significantly decreased the S100A4-mediated upregulation of GFAP and C3, along with the protein levels of p-NF-κB p65 and TLR4 (D). Quanti tative analysis confirmed these results, as shown in E and F. Immunofluorescence staining confirmed that, after confluence, adherent astrocytes activated by S100A4 presented significantly greater fluorescence intensities of both GFAP and C3 than did control cells. PDTC ammonium or Resatorvid treatment reduced the fluorescence intensity of GFAP and C3 (G) (scale bar, 100 μm). Quantitative analysis further confirmed these results, as shown in H. Data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 normal versus S100A4. #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001 S100A4 versus S100A4 + PDTC ammonium or Resatorvid

Article Snippet: Antibodies against S100A4 (16105-1-AP), C3 (21337- 1-AP), NF-κB p65 (10745-1-AP), and TLR4 (19811-1- AP) were purchased from Proteintech (Hubei, Wuhan, China).

Techniques: Activation Assay, In Vitro, Western Blot, Recombinant, Expressing, Immunofluorescence, Staining, Fluorescence, Control

Journal: The journal of headache and pain

Article Title: Inhibition of S100A4 decreases neurotoxic astrocyte reactivity and attenuates neuropathic pain via the TLR4/NF-κB pathway in a rat model of spinal nerve ligation.

doi: 10.1186/s10194-025-02045-9

Figure Lengend Snippet: Fig. 6 SNL upregulates spinal TLR4/NF-κB signalling, whereas S100A4 inhibition attenuates A1 astrocyte activation via this pathway. Western blot show ing the time courses of TLR4 and p-NF-κB p65 expression in sham and SNL rats (A). SNL induced a progressive increase in TLR4 and p-NF-κB p65 levels in the spinal cord, especially during the maintenance period of NP, and then tended to decrease from Days 1 to 21 after surgery (B). The data are presented as the means ± SEMs. For TLR4, ***p < 0.001, ****p < 0.0001 SNL versus sham. For NF-κB, ##p < 0.01, ###p < 0.001, ####p < 0.0001 SNL versus sham. Dual- label fluorescence colocalization analysis revealed that the intensity of GFAP/C3 double-positive fluorescence in the ipsilateral L4/5 SDH was significantly greater on POD 10 after SNL than in the sham group (scale bar, 500 μm). Immunofluorescence images (C1-C, C2-C and C3-C) revealed intense colo calization of p-NF-κB p65 (green) with NeuN (red) and limited coexpression with GFAP (red) but not with iba-1 (red) (scale bar, 500 μm). More details are shown at higher magnification in C1-D, C2-D and C3-D (scale bar, 100 μm). WB analysis revealed that successive intrathecal niclosamide injections beginning at 1 dpi induced significantly lower levels of TLR4 and p-NF-κB p65 expression at 10 and 14 days in SNL rats (D and E). The data are presented as the means ± SEMs. ***p < 0.001, ****p < 0.0001 SNL versus sham. ##p < 0.01, ###p < 0.001 SNL versus SNL + niclosamide. Moreover, the administration of Resatorvid caused a decrease in GFAP and C3 expression, as well as p-NF-κB p65 protein levels, at 10 and 14 days in SNL rats (F and G). Repeated ad ministration of the NF-κB inhibitor PDTC decreased the expression of GFAP and C3 (H and I). The data are presented as the means ± SEMs. ****p < 0.0001 sham versus SNL + DMSO. ###p < 0.001, ####p < 0.0001 SNL + DMSO versus SNL + PDTC ammonium or Resatorvid. Dual-label fluorescence colocalization analysis revealed that the intensity of GFAP/C3 double-positive fluorescence in the ipsilateral L4/5 SDH was significantly greater on POD 10 after SNL than in the sham group. Repeated administration of PDTC ammonium or Resatorvid decreased the intensity of GFAP+C3+ fluorescence (J and K) (scale bar, 500 μm). Quantitative analysis further confirmed these results, as shown in L and M. Data are presented as the mean ± SEM. ****p < 0.0001 sham versus SNL + DMSO. ###p < 0.001 SNL versus SNL + PDTC ammonium or Resatorvid

Article Snippet: Antibodies against S100A4 (16105-1-AP), C3 (21337- 1-AP), NF-κB p65 (10745-1-AP), and TLR4 (19811-1- AP) were purchased from Proteintech (Hubei, Wuhan, China).

Techniques: Inhibition, Activation Assay, Western Blot, Expressing, Fluorescence, Immunofluorescence

Journal: The journal of headache and pain

Article Title: Inhibition of S100A4 decreases neurotoxic astrocyte reactivity and attenuates neuropathic pain via the TLR4/NF-κB pathway in a rat model of spinal nerve ligation.

doi: 10.1186/s10194-025-02045-9

Figure Lengend Snippet: Fig. 7 Blocking TLR4/NF-κB signalling alleviates neuropathic pain after SNL and impacts exogenous S100A4-induced hyperalgesia. Repeated administra tion of PDTC ammonium (50 µg/20 µl) in the early postoperative phase (POD 1) partially attenuated the development of mechanical hypersensitivity in the ipsilateral hind paw of SNL rats (A), which was maintained until POD 14. Moreover, after receiving repeated intrathecal injections of 20 µg Resatorvid, the PWT of the ipsilateral hind paw was significantly greater than that of the saline group from Day 3 to Day 14 after SNL (A). Nonetheless, the treatment did not influence the PWT of the contralateral hind paws (B). The data are presented as the means ± SEMs. ****p < 0.0001 SNL + DMSO versus SNL + PDTC ammonium. ####p < 0.0001 SNL + DMSO versus SNL + Resatorvid. Moreover, pain behavioural assessment revealed that PDTC ammonium and Resator vid could partially reverse S100A4-induced mechanical hyperalgesia in the bilateral hind paws of naive rats, with the effect lasting up to 5 days (left, C; right, D). The data are presented as the means ± SEMs. ****p < 0.0001, saline versus S100A4. ####p < 0.0001 S100A4 versus S100A4 + PDTC ammonium. ѱѱѱp < 0.001, ѱѱѱѱp < 0.0001 S100A4 versus S100A4 + Resatorvid

Article Snippet: Antibodies against S100A4 (16105-1-AP), C3 (21337- 1-AP), NF-κB p65 (10745-1-AP), and TLR4 (19811-1- AP) were purchased from Proteintech (Hubei, Wuhan, China).

Techniques: Blocking Assay, Saline