Journal: Oncology Reports

Article Title: Platelets and MMP?9 contribute to esophageal cancer invasion via CD40?CD154 interactions

doi: 10.3892/or.2025.8912

Figure Lengend Snippet: Functional analysis of cells stimulated with CD154. (A) Viability assay. No significant changes were observed in all four cell lines. (B) Migration assay. (C) Invasion assay. Migration and invasion were enhanced in TE-5 and TE-10 cells, which highly expressed CD40, after rsCD154 stimulation. No significant changes were observed in low CD40 expression cells (TE-4 and −11 cells). The Mann-Whitney U test was performed using GraphPad Prism 9 software. ns, not significant; OD, optical density; rsCD154, recombinant soluble CD154.

Article Snippet: CD154 , REA238 , FC , 1:50 , Miltenyi Biotec GmbH.

Techniques: Functional Assay, Viability Assay, Migration, Invasion Assay, Expressing, MANN-WHITNEY, Software, Recombinant

Journal: Oncology Reports

Article Title: Platelets and MMP?9 contribute to esophageal cancer invasion via CD40?CD154 interactions

doi: 10.3892/or.2025.8912

Figure Lengend Snippet: Changes in gene expression caused by the CD40-CD154 interaction in TE-10 cells. Screening of 40 genes involved in extracellular matrix remodeling. The expression ratios of target genes relative to GAPDH are shown on the left. On the right, CD154-induced changes in gene expression are displayed in descending order. Upregulation of gene expression in the order MMP-9 > PLG > LAMC2 was confirmed following rsCD154 stimulation.

Article Snippet: CD154 , REA238 , FC , 1:50 , Miltenyi Biotec GmbH.

Techniques: Gene Expression, Expressing

Journal: Oncology Reports

Article Title: Platelets and MMP?9 contribute to esophageal cancer invasion via CD40?CD154 interactions

doi: 10.3892/or.2025.8912

Figure Lengend Snippet: Changes in MMP-9 expression caused by the CD40-CD154 interaction in TE cells. (A) Qualitative analysis of MMP-9 secretion from TE cells using gelatin zymography. Increased MMP-9 secretion was observed in high CD40 expression cells (TE-5 and −10), regardless of the baseline MMP-9 secretion level without CD154 stimulation. (B) Quantitative analysis of MMP-9 secretion from TE cells using an ELISA. Increased MMP-9 secretion was observed in TE-5 and −10 cells. (C) Quantitative analysis of MMP-9 mRNA expression in TE cells via reverse transcription-quantitative PCR. MMP-9 mRNA expression upregulation was observed in high CD40 expression cells (TE-5 and −10). The Mann-Whitney U test was performed using GraphPad Prism 9 software. n.d., not detected; rsCD154, recombinant soluble CD154.

Article Snippet: CD154 , REA238 , FC , 1:50 , Miltenyi Biotec GmbH.

Techniques: Expressing, Zymography, Enzyme-linked Immunosorbent Assay, Reverse Transcription, Real-time Polymerase Chain Reaction, MANN-WHITNEY, Software, Recombinant

Journal: Oncology Reports

Article Title: Platelets and MMP?9 contribute to esophageal cancer invasion via CD40?CD154 interactions

doi: 10.3892/or.2025.8912

Figure Lengend Snippet: Experiments using CD40-knockdown TE-10 cells using siRNA. (A) Transfection with CD40 siRNA resulted in a reduction in CD40 mRNA expression. The average relative CD40 mRNA expression was compared using GraphPad Prism 9 software. After one-way ANOVA, Bonferroni post hoc analysis was performed. (B) CD154 stimulation of CD40 knockdown TE-10 cells was performed, and MMP-9 upregulation was suppressed. (C) Migration assay. CD40 knockdown reduced cell migration, and no increase in migration was observed following CD154 stimulation. The images on the right present a visualization of migrating cells under a 1.25× optical microscope, with the cellular regions indicated by green labelling. (D) Invasion assay. Similar to the migration assay, a decrease in invasion was observed following CD40 knockdown. The images on the right present a visualization of migrating cells under a 1.25× optical microscope, with the cellular regions indicated by green labelling. siRNA, small interfering RNA.

Article Snippet: CD154 , REA238 , FC , 1:50 , Miltenyi Biotec GmbH.

Techniques: Knockdown, Transfection, Expressing, Software, Migration, Microscopy, Invasion Assay, Small Interfering RNA

Journal: Oncology Reports

Article Title: Platelets and MMP?9 contribute to esophageal cancer invasion via CD40?CD154 interactions

doi: 10.3892/or.2025.8912

Figure Lengend Snippet: Survival analysis of patients who underwent esophagectomy was performed using the Kaplan-Meier method. The log-rank (Mantel-Cox) test was conducted using GraphPad Prism 9 software. (A) Association between DFS and MMP-9 levels. (B) Association between DFS and CD154 levels. (C) Association between OS and MMP-9 levels. (D) Association between OS and CD154 levels. DFS, disease-free survival; MST, median survival time; OS, overall survival.

Article Snippet: CD154 , REA238 , FC , 1:50 , Miltenyi Biotec GmbH.

Techniques: Software

Journal: Oncology Reports

Article Title: Platelets and MMP?9 contribute to esophageal cancer invasion via CD40?CD154 interactions

doi: 10.3892/or.2025.8912

Figure Lengend Snippet: Survival analysis of patients with esophageal cancer stratified by disease stage. (A) Association between DFS and MMP-9 levels in patients with pathological stage I. (B) Association between DFS and CD154 levels in patients with pathological stage I. (C) Association between OS and MMP-9 levels in patients with pathological stage I. (D) Association between OS and CD154 levels in patients with pathological stage I. (E) Association between DFS and MMP-9 levels in patients with pathological stage II–IV. (F) Association between DFS and CD154 levels in patients with pathological stage II–IV. (G) Association between OS and MMP-9 levels in patients with pathological stage II–IV. (H) Association between OS and CD154 levels in patients with pathological stage II–IV. Survival analysis was performed using the Kaplan-Meier method, and the log-rank (Mantel-Cox) test was conducted using GraphPad Prism 9 software. DFS, disease-free survival; MST, median survival time; OS, overall survival.

Article Snippet: CD154 , REA238 , FC , 1:50 , Miltenyi Biotec GmbH.

Techniques: Software

Journal: Oncology Reports

Article Title: Platelets and MMP?9 contribute to esophageal cancer invasion via CD40?CD154 interactions

doi: 10.3892/or.2025.8912

Figure Lengend Snippet: Schema of the CD40-CD154 interaction in the tumor microenvironment of esophageal cancer. In tumor cells, the CD40-CD154 interaction induces the secretion of molecules involved in ECM remodeling, including MMP-9, contributing to cell invasion, EMT and metastasis. In immune cells, the CD40-CD154 interaction is involved in antitumor immunity, leading to cancer cell death. ECM, extracellular matrix; EMT, epithelial-mesenchymal transition.

Article Snippet: CD154 , REA238 , FC , 1:50 , Miltenyi Biotec GmbH.

Techniques:

Journal: PLOS Pathogens

Article Title: Regulatory T cell-like response to SARS-CoV-2 in Jamaican fruit bats ( Artibeus jamaicensis ) transduced with human ACE2

doi: 10.1371/journal.ppat.1011728

Figure Lengend Snippet: Four bats were transduced with human ACE2 and infected 5 days later. After 12 days, splenocytes were recovered for AIM testing and gene expression analsysis. A. Splenocytes from hACE2-transduced bats 12 days post infection with SARS-CoV were cultured with or without SARS-CoV-2 nucleocapsid peptide library for 6 hours in the presence of anti-CD40 blocking antibody (AIM testing) or 24 hours without anti-CD40 blocking antibody (cellular RNA). B. Cells were stained with anti-CD154 and anti-CD4 and analyzed by flow cytometry. Violin plots for the individual bats, plus the pooled sample and rank data. In the presence of peptide, CD154 expression was significantly increased on splenocytes (red) compared to no peptide controls (blue) (Wilcoxon rank sum test). C. Heat map of splenocyte qPCR (ΔΔCq) gene expression in response to peptide stimulation determined 3 of the 4 bats expressed a profile consistent with a regulatory T cell response. D. Peptide-induced gene expression changes in splenocyte cultures of bats 1, 2 and 4 from panel C; IL-10, TGFβ and CD4 gene expression were significantly elevated, whereas CXCR4 was nearly significant (two-tailed t-test).

Article Snippet: Based upon structural homology , we determined that commercially available anti-mouse CD40 (Tonbo, clone FGK45) and anti-human CD154 (Tonbo, clone 5C8) monoclonal antibodies were cross reactive with Jamaican fruit bat orthologs ( ).

Techniques: Transduction, Infection, Gene Expression, Cell Culture, Blocking Assay, Staining, Flow Cytometry, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: CBP/EP300 acetylates and stabilizes the stress-responsive Heat Shock Factor 2, a process compromised in Rubinstein-Taybi syndrome

doi: 10.1101/481457

Figure Lengend Snippet: (A) The ectopically expressed YFP-HSF2 protein is acetylated by exogenous HA-CBP or EP300. Representative immunoblots (n=5 independent experiments). HEK 293 cells were transfected with different combinations of YFP-HSF2, HA-CBP, HA-EP300 constructs, and mock-HA or -GFP constructs. YFP-HSF2 was immunoprecipitated using anti-GFP-trap antibody (IP GFP-Trap) and its acetylation status was determined by WB analyses, using an anti-pan-acetyl-lysine antibody (AcK; left panels). Immunoprecipitated HSF2 was detected using an anti-GFP antibody (WB: GFP). The total amounts of the proteins in the input samples were detected with anti-GFP or anti-HA antibodies (inputs; right panels). Actin, loading control. C TA :Trap®-A beads were used as a negative control (see Experimental Procedures). (B) The HSF2-Myc protein is not acetylated by a dominant-negative form of CBP (DNCBP-HA). HEK 293 cells were transfected as in ( A ), except that HSF2-Myc was used instead of HSF2-YFP, and immunoprecipitation was performed using anti-Myc-trap antibody (IP Myc-Trap). Representative immunoblots (n=2 experiments). C TA : Trap®-A beads were used as a negative control (see Experimental Procedures). HSP90: loading control. (C) Schematic representation of the eight main acetylated lysine residues of the HSF2 protein. Purified mouse Flag-HSF2, co-expressed with HA-EP300, immunoprecipitated and subjected to MS analysis for detection of acetylated lysine residues. The three lysine residues K128, K135, K197, located in the oligomerization domain (HR-A/B), are enlightened in red and K82, located in the DBD, in blue; the other four lysine residues (K209/K210, K395/K401) are indicated in regular black. The DNA-binding domain (DBD, orange); the oligomerization domain (HR-A/B; green) and the domain controlling oligomerization (the leucine-zipper-containing HR-C; green); as well as the N-terminal domain (activation domain TAD; red) are illustrated. The boundaries of each domain are indicated in bold and blue. Bold and blue numbers correspond to the number of the amino acids located at boundaries of the domains of the mouse HSF2 protein, numbered from the +1 (ATG); the equivalent in the human HSF2 protein, if different, are indicated in bold and black. These four (K82, K128, K135, K197) or three lysine residues (K128, K135, K197) were mutated into glutamines (4KQ or 3KQ, respectively) or arginines (4KR or 3KR, respectively; see also Figure S2A). (D) The mutations of three or four lysine (K) residues to arginine (3KR or 4KR) or glutamine residues (3KQ or 4KQ) decrease global HSF2 acetylation levels. HEK 293 cells were co-transfected with EP300-HA and wild-type (WT) or mutated human HSF2-Myc on the indicated lysine residues. After immunoprecipitation of HSF2, using anti-Myc antibody, its acetylation was analysed by WB using an anti-AcK antibody. HSC70, loading control. n=3 independent experiments. (See also Figure S2B,C). (E) In vitro acetylation of HSF2 peptides containing the K135 or K197 residues by recombinant CBP Full-HAT . Time course of reverse phase-ultra-fast liquid chromatography (RP-UFLC) analysis of the acetylation of HSF2K197 (upper panel) and HSF2K135 peptides (lower panel) by CBP Full-HAT. Aliquots of the reaction were collected at 0 (black), 1 (red) or 2 (green) hours and elution of peptides was monitored by fluorescence emission at 530 nm (excitation: 485 nm, uV: arbitrary unit of fluorescence; see Figure S2D for HSF2K82 peptide). (F) Quantification of the in vitro acetylated HSF2 peptides containing K82, K135, and K197 residues. The AUC (area under the curve) of the acetylated K82, K135 and K197 peptides was quantified and converted in product concentration using a calibrated curve of various known concentrations of peptides. Note that it was not possible to investigate the acetylation of the HSF2 K128 peptide by CBP, because this peptide was repeatedly insoluble at the synthesis steps (Manufacturer’s information; see also Figure S2D-F).

Article Snippet: Cells were lysed in Lysis buffer (50 mM Hepes pH 8, 100 mM NaCl, 5 mM EDTA, Triton X-100 0.5 %, Glycerol 10 %, VPA (1 mM), DTT 1 mM, PMSF 1 mM, proteases inhibitors, phosphatase inhibitors (Roche)) and then, HSF2 was immunoprecipitated using anti-GFP- or anti-Myc-trap antibody, or as a control Trap®-A control (ChromoTek).

Techniques: Western Blot, Transfection, Construct, Immunoprecipitation, Negative Control, Dominant Negative Mutation, Purification, Binding Assay, Activation Assay, In Vitro, Recombinant, Liquid Chromatography, Fluorescence, Concentration Assay