quantikine elisa  (R&D Systems)

Journal: Stem Cell Research & Therapy

Article Title: IL-1β modulates inflammatory response of human bone marrow-derived MSCs and neutrophil recruitment in vitro via NF-kB-associated signaling

doi: 10.1186/s13287-026-05029-x

Figure Lengend Snippet: IL-1β stimulation increases expression of neutrophil recruitment and NF-kB signaling genes by BM-hMSCs. Heatmap clustering of the Z-score of the top 20 differentially expressed genes between unstimulated control BM-hMSCs (3 replicates, 1 donor, green) and IL-1 β stimulated BM-hMSCs (3 replicates, 1 donor, purple). Red color indicates higher expression of the genes and blue color indicates decreased expression A . Protein expression of CXCL1 (10 replicates, 4 donors), CCL2 (4 replicates, 2 donors), CXCL5 (4 replicates, 2 donors), and CXCL8/IL-8 (7 replicates, 3 donors) in BM-hMSCs secretome measured by ELLA or ELISA. Data are presented as median, and statistical analysis was performed using unpaired t-test with Welch’s correction B . Bar plot of the top 20 significant Gene Ontology (GO) biological processes representing IL- 1β induced upregulated differentially expressed genes C IL-1 β, Interleukin-1β; BM-hMSCs, Bone marrow derived human mesenchymal cells; CCL2, chemokine (C–C motif) ligand 2; CXCL1, chemokine (C-X-C motif) ligand 1; CXCL2, chemokine (C-X-C motif) ligand 1; CXCL8, chemokine (C-X-C motif) ligand 8; ****, p < 0,0001

Article Snippet: CXCL1 was measured in diluted 1:1 or undiluted CM samples using Quantikine ELISA (cat# DGR00B, R&D Systems).

Techniques: Expressing, Control, Enzyme-linked Immunosorbent Assay, Derivative Assay

Journal: Radiation Oncology (London, England)

Article Title: CXCR2 affects sensitization of radioresistant HPV-negative head and neck squamous cell carcinoma cells by ABT-263

doi: 10.1186/s13014-026-02798-w

Figure Lengend Snippet: ABT-263 decreases senescence and alters the SASP in UPCI:SCC040 and Cal33 cells. Cells were treated with 1 µM ABT-263 (Cal33) or 5 µM of ABT-263 (UPCI:SCC040) concomitantly with 6 Gy irradiation and analyzed on D4 or D6. ( A-B ) Gene expression of secreted factors measured by qRT-PCR and normalized to unirradiated DMSO controls. ( A ) UPCI:SCC040. ( B ) Cal33. ( C-D ) Secreted IL1A, IL1B, IL8 and CXCL1 protein levels were detected by ELISA on D4 and D6. Protein concentrations (pg/ml) were normalized to cell number. ( C ) UPCI:SCC040. ( D ) Cal33. ( E-F ) Relative CXCR2 gene expression shown as ΔCT values in ( E ) Cal33 and ( F ) UPCI:SCC040 cells. Values a represent means ± SEM of N = 2. Student’s t test: p < 0.05 (*), p < 0.01 (**), ns: not significant

Article Snippet: Cytokines IL-1α, IL-1β, IL-8 and CXCL1 were measured using DuoSet ELISA kits (R&D Systems, IL-1α Cat#DY200, IL-1β Cat#DY201-05, IL8 Cat#30021151, CXCL1 Cat#DY 275) according to manufacturer instructions.

Techniques: Irradiation, Gene Expression, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: Histone lactylation increases CXCL1 expression for neutrophil infiltration and immune escape in pancreatic cancer

doi: 10.1038/s41467-026-69311-5

Figure Lengend Snippet: A The flowchart illustrates that RNA sequencing was conducted using two PDAC cell lines with or without 2DG treatment. Subsequently, the intersection of detected genes within the chemokine family in both cell lines is identified, and a heatmap is generated to display the log 2 fold change (2DG versus vehicle) of these genes. B , C Relative mRNA levels of Cxcl1 in PDAC cell lines following glycolysis inhibition, either by using 2DG or silencing LDH, were analyzed by qRT-PCR ( n = 3 independent experiments). D , E Relative protein levels of CXCL1 in PDAC cell lines following glycolysis inhibition, either by using 2DG or silencing LDH, were determined using ELISA assay ( n = 3 independent experiments). F Representative IHC images of subcutaneous tumors ( n = 6 mice for 2DG treatment, n = 5 mice for shLDH/shNTC groups) stained by CXCL1 antibody (scale bars = 100 μm). G Relative serum CXCL1 levels in mice treated with or without 2DG were measured by ELISA ( n = 6 mice per group). H Relative serum CXCL1 levels in healthy donors and PDAC patients were measured by ELISA (22 healthy samples and 27 PDAC samples). I Schematic diagram showing the in vitro migration assay: human/mouse neutrophils were co-incubated with the culture medium supernatant from PDAC cells treated with 2DG or LDH-knockdown. J Neutrophils were co-cultured with CD8 + T cells in different proportions, and the proliferation of CD8 + T cells was detected with the CFSE assay ( n = 6 biologically independent samples). K The migratory activity of neutrophils co-incubated with the culture medium supernatant from PDAC cells treated with shLDH and recombinant CXCL1 was analyzed by counting the penetrated cell numbers ( n = 3 biologically independent samples). L The neutrophils were treated with SX-682 or Navarixin for 1.5 h in advance, and the migratory activity of neutrophils co-incubated with the culture medium supernatant from PDAC cell lines treated with shLDH was analyzed by counting the penetrated cell numbers ( n = 3 biologically independent samples). M Representative luminescence images of the orthotopic tumor in mice and statistical analysis of MFI ( n = 5 mice per group). N Tumor-infiltrating neutrophils isolated from the orthotopic tumors were analyzed using flow cytometry ( n = 5 mice per group). Data represent mean ± SEM. Statistical analysis was conducted using the two-tailed unpaired Student’s t test. Source data are provided as a Source Data file.

Article Snippet: The primary antibodies used here included anti-LDHA antibody (Proteintech, #19987-1-AP, 1:3000), anti-LDHB antibody (Proteintech, #14824-1-AP, 1:5000), anti-H3 antibody (Proteintech, #17168-1-Ap, 1:3000), anti-PCAF antibody (Cell Signaling Technology, #3378S, 1:1000), anti-P300 antibody (Proteintech, #20695-1-AP, 1:500), anti-GCN5 antibody (Proteintech, #66575-1-Ig, 1:3000), anti-beta Actin antibody (PTMBIO, #PTM-5028, 1:1000), anti-human CXCL1 Polyclonal antibody (Proteintech, #12335-1-AP), anti-mouse CXCL1 Polyclonal antibody (Proteintech, #30322-1-AP), anti-L-Lactyl Lysine antibody (PTMBIO, #PTM-1401RM, 1:1000), anti-L-Lactyl-Histone H3K18 antibody (PTMBIO, #PTM-1406RM, 1:1000), anti-L-Lactyl-Histone H3K9 antibody (PTMBIO, #PTM-1419RM, 1:1000), anti-L-Lactyl-Histone H3K14 antibody (PTMBIO, #PTM-1414RM, 1:1000), anti-H4 antibody (PTMBIO, #PTM-1009, 1:1000), anti-L-Lactyl-Histone H4K12 antibody (PTMBIO, #PTM-1411RM, 1:1000), anti-L-Lactyl-Histone H4K8 antibody (PTMBIO, #PTM-1415RM, 1:1000), anti-Acetyl-Histone H3 (Lys18) antibody (PTMBIO, #PTM-114RM, 1:1000), anti-Acetyllysine antibody (PTMBIO, #PTM-101, 1:1000), and anti-Acetyl-Histone H4 (Lys8) antibody (PTMBIO, #PTM-190, 1:1000).

Techniques: RNA Sequencing, Generated, Inhibition, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Staining, In Vitro, Migration, Incubation, Knockdown, Cell Culture, CFSE Assay, Activity Assay, Recombinant, Isolation, Flow Cytometry, Two Tailed Test

Journal: Nature Communications

Article Title: Histone lactylation increases CXCL1 expression for neutrophil infiltration and immune escape in pancreatic cancer

doi: 10.1038/s41467-026-69311-5

Figure Lengend Snippet: A Lactylation levels of pan-lysine and different lysine sites in histone H3 of 7 pairs of PDAC tissue and adjacent normal tissue were detected by western blot. B Representative IHC images of clinical PDAC and adjacent normal tissue stained by H3K18la antibody ( n = 79 patients). C The diagram illustrating the targets of 2DG, shRNA, and rotenone in the glycolysis process. D , E Relative levels of H3K18la and PanKla in PDAC cells treated with gradient doses of 2DG ( D ) or rotenone ( E ) for 24 h were detected by western blot. F Relative levels of H3K18la and PanKla in subcutaneous tumors treated with or without 2DG were detected by western blot. ( n = 4 biologically independent samples per group). G H3K18la levels of subcutaneous tumors treated with or without 2DG were detected by immunofluorescence staining. H The H3K18la levels in PDAC cells treated with 2DG or sodium lactate were detected by western blot. I The H3K18la level in PANC1 and PANC02 cells following LDH-knockdown and sodium lactate treatment by western blot. J Schematic diagram of the CUT&Tag assay for PanKla or H3K18la in BxPC3 cells with or without 2DG treatment. K TSS heatmaps presenting the binding density of PanKla and H3K18la with or without 2DG treatment. L Venn diagram indicating the overlap of differentially expressed genes in TCGA-PAAD between PDAC and adjacent normal tissue, differentially expressed genes in RNA-seq after 2DG treatment, and differentially bound genes of H3K18la and PanKla after 2DG treatment. M Genome browser track analysis shows the PanKla and H3K18la levels in the Cxcl1 promoter region with or without 2DG treatments. N The Cxcl1 promoters that are bound by H3K18la and PanKla in PDAC cells were quantified using ChIP-qPCR assays ( n = 3 independent experiments). O Relative mRNA levels of Cxcl1 in PDAC cells treated with 2DG or sodium lactate were detected by qRT-PCR ( n = 3 independent experiments). P Representative IHC images stained by CXCL1 or H3K18la antibody and correlation analysis of H-scores of CXCL1 and H3K18la in PDAC samples ( n = 21 patients). Scale bar = 100 μm. Data represent mean ± SEM. Statistical analysis was conducted using the two-tailed unpaired Student’s t test ( N–O ) and two-tailed Pearson’s correlation analysis ( P ). Unless otherwise indicated, all western blots had three independent experimental repetitions with consistent results, where Histone H3, H3K9la, H3K14la, H3K18la, and PanKla blots are from parallel-processed separate gels (size conflict) with samples from the same experiment. Source data are provided as a Source Data file.

Article Snippet: The primary antibodies used here included anti-LDHA antibody (Proteintech, #19987-1-AP, 1:3000), anti-LDHB antibody (Proteintech, #14824-1-AP, 1:5000), anti-H3 antibody (Proteintech, #17168-1-Ap, 1:3000), anti-PCAF antibody (Cell Signaling Technology, #3378S, 1:1000), anti-P300 antibody (Proteintech, #20695-1-AP, 1:500), anti-GCN5 antibody (Proteintech, #66575-1-Ig, 1:3000), anti-beta Actin antibody (PTMBIO, #PTM-5028, 1:1000), anti-human CXCL1 Polyclonal antibody (Proteintech, #12335-1-AP), anti-mouse CXCL1 Polyclonal antibody (Proteintech, #30322-1-AP), anti-L-Lactyl Lysine antibody (PTMBIO, #PTM-1401RM, 1:1000), anti-L-Lactyl-Histone H3K18 antibody (PTMBIO, #PTM-1406RM, 1:1000), anti-L-Lactyl-Histone H3K9 antibody (PTMBIO, #PTM-1419RM, 1:1000), anti-L-Lactyl-Histone H3K14 antibody (PTMBIO, #PTM-1414RM, 1:1000), anti-H4 antibody (PTMBIO, #PTM-1009, 1:1000), anti-L-Lactyl-Histone H4K12 antibody (PTMBIO, #PTM-1411RM, 1:1000), anti-L-Lactyl-Histone H4K8 antibody (PTMBIO, #PTM-1415RM, 1:1000), anti-Acetyl-Histone H3 (Lys18) antibody (PTMBIO, #PTM-114RM, 1:1000), anti-Acetyllysine antibody (PTMBIO, #PTM-101, 1:1000), and anti-Acetyl-Histone H4 (Lys8) antibody (PTMBIO, #PTM-190, 1:1000).

Techniques: Western Blot, Staining, shRNA, Immunofluorescence, Knockdown, Binding Assay, RNA Sequencing, ChIP-qPCR, Quantitative RT-PCR, Two Tailed Test

Journal: Nature Communications

Article Title: Histone lactylation increases CXCL1 expression for neutrophil infiltration and immune escape in pancreatic cancer

doi: 10.1038/s41467-026-69311-5

Figure Lengend Snippet: A Molecular docking simulated the binding affinities of PCAF for acetyl-CoA and lactyl-CoA. B Co-IP was performed to confirm the interaction between H3K18la and PCAF in PANC1 and KPC cells. PCAF and H3K18la were detected on the same gel. C, D Western blot analysis demonstrates the level of H3K18la in PDAC cells with or without the treatment of PCAF inhibitors (bromosporine or Embelin). Histone H3 and H3K18la blots are from parallel-processed separate gels (size conflict) with samples from the same experiment. E Western blot analysis demonstrates the level of H3K18la in PDAC cells following the knockdown of Pcaf . Histone H3, PCAF, β-actin and H3K18la blots are from parallel-processed separate gels with samples from the same experiment. F Western blots of in vitro histone acetylation or lactylation assay, the samples as indicated. Blots are from parallel-processed separate gels with samples from the same experiment. G , H Relative Cxcl1 RNA and protein levels with the vehicle or bromosporine treatment were analyzed by qRT-PCR and ELISA ( n = 3 independent experiments), respectively. I Relative Cxcl1 RNA levels after Pcaf knockdown were analyzed by qRT-PCR ( n = 3 independent experiments). J A schematic diagram showing the orthotopic tumor construction in C57BL/6 J mice ( n = 5 mice per group in one experiment, 2 × 10 6 KPC-luc cells per mouse) with or without bromosporine treatment. K , L Image and weights of the orthotopic tumors in the experimental groups from ( J ) at the end of the experiments ( n = 5 mice per group). M Lactylation levels of H3K18 and PanK in orthotopic tumors with or without bromosporine treatment were detected by western blot ( n = 4 biologically independent samples per group). Histone H3, H3K18la, and PanKla blots are from parallel-processed separate gels (size conflict) with samples from the same experiment. N Serum CXCL1 levels in mice with or without bromosporine treatment were measured by ELISA ( n = 5 mice per group). O – R Tumor-infiltrating neutrophils, CD8 + T cells, GZMB + CD8 + T cells, and PD-1 + CD8 + T cells isolated from the orthotopic tumors were analyzed using flow cytometry. Representative graphs of flow cytometry (left panel) and statistical analysis of the cell ratio (right panel) ( n = 5 mice per group). S The migratory abilities of neutrophils co-incubated with the culture medium supernatant from shNTC/ Pcaf PDAC cells treated with recombinant CXCL1 were analyzed by counting the penetrated cell numbers ( n = 3 biologically independent samples). T Representative IHC images stained by CXCL1 or H3K18la antibody (scale bar = 100 μm, left panel), correlation analysis of H-scores of CXCL1 and H3K18la in PDAC samples ( n = 21 patients, right panel). Data represent mean ± SEM. Statistical analysis was conducted using the two-tailed unpaired Student’s t test ( G – I , L , and N – S ) and two-tailed Pearson’s correlation analysis ( T ). Unless otherwise indicated, all western blots had three independent experimental repetitions with consistent results. Source data are provided as a Source Data file.

Article Snippet: The primary antibodies used here included anti-LDHA antibody (Proteintech, #19987-1-AP, 1:3000), anti-LDHB antibody (Proteintech, #14824-1-AP, 1:5000), anti-H3 antibody (Proteintech, #17168-1-Ap, 1:3000), anti-PCAF antibody (Cell Signaling Technology, #3378S, 1:1000), anti-P300 antibody (Proteintech, #20695-1-AP, 1:500), anti-GCN5 antibody (Proteintech, #66575-1-Ig, 1:3000), anti-beta Actin antibody (PTMBIO, #PTM-5028, 1:1000), anti-human CXCL1 Polyclonal antibody (Proteintech, #12335-1-AP), anti-mouse CXCL1 Polyclonal antibody (Proteintech, #30322-1-AP), anti-L-Lactyl Lysine antibody (PTMBIO, #PTM-1401RM, 1:1000), anti-L-Lactyl-Histone H3K18 antibody (PTMBIO, #PTM-1406RM, 1:1000), anti-L-Lactyl-Histone H3K9 antibody (PTMBIO, #PTM-1419RM, 1:1000), anti-L-Lactyl-Histone H3K14 antibody (PTMBIO, #PTM-1414RM, 1:1000), anti-H4 antibody (PTMBIO, #PTM-1009, 1:1000), anti-L-Lactyl-Histone H4K12 antibody (PTMBIO, #PTM-1411RM, 1:1000), anti-L-Lactyl-Histone H4K8 antibody (PTMBIO, #PTM-1415RM, 1:1000), anti-Acetyl-Histone H3 (Lys18) antibody (PTMBIO, #PTM-114RM, 1:1000), anti-Acetyllysine antibody (PTMBIO, #PTM-101, 1:1000), and anti-Acetyl-Histone H4 (Lys8) antibody (PTMBIO, #PTM-190, 1:1000).

Techniques: Binding Assay, Co-Immunoprecipitation Assay, Western Blot, Knockdown, In Vitro, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Isolation, Flow Cytometry, Incubation, Recombinant, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: Histone lactylation increases CXCL1 expression for neutrophil infiltration and immune escape in pancreatic cancer

doi: 10.1038/s41467-026-69311-5

Figure Lengend Snippet: A A schematic diagram showing the subcutaneous tumor model ( n = 6 mice per group in one experiment, 6 × 10 6 PANC02 cells per mouse) treated with bromosporine and anti-PD-1 antibody. B. The tumor volume growth curves of subcutaneous tumors from ( A ). C , D Image and weights of the subcutaneous tumors at the end point of experiments ( n = 6 mice per group). E The statistical analysis of the cell ratio of tumor-infiltrating neutrophils and CD8 + T cells isolated from subcutaneous tumors ( n = 3 mice per group). F Western blot analysis demonstrates H3K18la levels in the subcutaneous tumors ( n = 3 biologically independent samples per group) from ( A ). Histone H3 and H3K18la blots are from parallel-processed separate gels (size conflict) with samples from the same experiment. G Relative serum CXCL1 levels in mice treated with or without bromosporine/anti-PD-1 antibody were measured by ELISA ( n = 6 mice per group). H A schematic diagram showing the combinational treatment schedule for the orthotopic KPC-luc tumor model ( n = 21 mice per group in one experiment, 2 × 10 6 KPC-luc cells per mouse). I , J Image and weights of the orthotopic tumors at the end point of experiments ( n = 6 mice per group). K – N Tumor-infiltrating neutrophils, CD8 + T cells, GZMB + CD8 + T cells, and PD-1 + CD8 + T cells were analyzed using flow cytometry. Representative graphs of flow cytometry (left panel) and statistical analysis of the cell ratio (right panel) ( n = 6 mice per group). O Western blot analysis showing the H3K18la levels in the orthotopic tumors ( n = 3 biologically independent samples per group) from ( H ). Histone H3 and H3K18la blots are from parallel-processed separate gels (size conflict) with samples from the same experiment. P Representative luminescence images and Quantification of radiance intensity of the mouse model in ( H ). Q Survival probability of mice with orthotopically transplanted PDAC ( n = 15 mice per group). R A working model displaying the signaling pathway through which the aerobic glycolysis-mediated Lactate-PCAF-H3K18la-CXCL1 axis modulates the tumor microenvironment in pancreatic cancer, and the scientific basis for the development of a novel therapeutic strategy for PDAC. Data represent mean ± SEM. Statistical analysis was conducted using the two-tailed unpaired Student’s t test ( B , D , E , G , J – N , P ) and the log-rank test ( Q ). Source data are provided as a Source Data file.

Article Snippet: The primary antibodies used here included anti-LDHA antibody (Proteintech, #19987-1-AP, 1:3000), anti-LDHB antibody (Proteintech, #14824-1-AP, 1:5000), anti-H3 antibody (Proteintech, #17168-1-Ap, 1:3000), anti-PCAF antibody (Cell Signaling Technology, #3378S, 1:1000), anti-P300 antibody (Proteintech, #20695-1-AP, 1:500), anti-GCN5 antibody (Proteintech, #66575-1-Ig, 1:3000), anti-beta Actin antibody (PTMBIO, #PTM-5028, 1:1000), anti-human CXCL1 Polyclonal antibody (Proteintech, #12335-1-AP), anti-mouse CXCL1 Polyclonal antibody (Proteintech, #30322-1-AP), anti-L-Lactyl Lysine antibody (PTMBIO, #PTM-1401RM, 1:1000), anti-L-Lactyl-Histone H3K18 antibody (PTMBIO, #PTM-1406RM, 1:1000), anti-L-Lactyl-Histone H3K9 antibody (PTMBIO, #PTM-1419RM, 1:1000), anti-L-Lactyl-Histone H3K14 antibody (PTMBIO, #PTM-1414RM, 1:1000), anti-H4 antibody (PTMBIO, #PTM-1009, 1:1000), anti-L-Lactyl-Histone H4K12 antibody (PTMBIO, #PTM-1411RM, 1:1000), anti-L-Lactyl-Histone H4K8 antibody (PTMBIO, #PTM-1415RM, 1:1000), anti-Acetyl-Histone H3 (Lys18) antibody (PTMBIO, #PTM-114RM, 1:1000), anti-Acetyllysine antibody (PTMBIO, #PTM-101, 1:1000), and anti-Acetyl-Histone H4 (Lys8) antibody (PTMBIO, #PTM-190, 1:1000).

Techniques: Isolation, Western Blot, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Two Tailed Test