Journal: Frontiers in Immunology

Article Title: CXCL2 affects macrophage antitumor function and immunotherapy efficacy in esophageal squamous cell carcinoma through calcium signaling

doi: 10.3389/fimmu.2026.1695387

Figure Lengend Snippet: ScRNA-seq analysis identified macrophage-specific genes associated with immunotherapy response in ESCC. (A) UMAP plot of single cells from patients with ESCC in GSE203115 cohort. (B) Heatmap of marker genes in each single cell subcluster based on the clustering analysis. (C, D) GO and KEGG analyses of differentially expressed genes in macrophages between the responsive and non-responsive groups. (E) Venn diagram of intersected gene in the indicated three signaling pathways. (F) CXCL2 expression levels on macrophages in the responding and non-responding groups. ESCC, esophageal squamous cell carcinoma; CXCL2, CXC chemokine ligand 2; GO, gene ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; UMAP, Uniform Manifold Approximation and Projection; scRNA-seq, single-cell RNA sequencing.

Article Snippet: Once tumors became palpable (approximately 5–7 days post-injection), the mice were randomly divided into groups (5 mice per group) and treated as follows: intraperitoneal injection of 100 μg CXCL2 recombinant protein (Cat. HY-P7258; MCE) once every two days, 150 μg anti-PD-1 antibody (Cat. BP0273; BioX Cell) once every three days , or the combination of both.

Techniques: Marker, Single Cell, Protein-Protein interactions, Expressing, RNA Sequencing

Journal: Frontiers in Immunology

Article Title: CXCL2 affects macrophage antitumor function and immunotherapy efficacy in esophageal squamous cell carcinoma through calcium signaling

doi: 10.3389/fimmu.2026.1695387

Figure Lengend Snippet: High infiltration of CXCL2 + macrophages is positively associated with favorable prognosis in ESCC patients. (A) Representative images of immunofluorescence co-staining of CD68 (red) and CXCL2 (green) in ESCC tissues. Scale bar, 20µm (left) and 5µm (right). (B) Pearson correlation analysis of CXCL2 expression level with the infiltration proportion of M1 or M2 macrophage in ESCC. (C) Kaplan-Meier curve for PFS of patients with low or high CXCL2 expression in TCGA cohort. Log-rank test. (D) Kaplan-Meier curve for OS of patients with low or high CXCL2 + macrophage population in our ESCC patient cohort. Log-rank test. (E) Forest plot illustrating the univariate and multivariate Cox proportional hazards regression models for OS in ESCC patients from our own cohort. ESCC, esophageal squamous cell carcinoma; CXCL2, CXC chemokine ligand 2; OS, overall survival; PFS, Progression-free survival; TNM, tumor-node-metastasis; HR, hazard ratio; CI, confidence interval.

Article Snippet: Once tumors became palpable (approximately 5–7 days post-injection), the mice were randomly divided into groups (5 mice per group) and treated as follows: intraperitoneal injection of 100 μg CXCL2 recombinant protein (Cat. HY-P7258; MCE) once every two days, 150 μg anti-PD-1 antibody (Cat. BP0273; BioX Cell) once every three days , or the combination of both.

Techniques: Immunofluorescence, Staining, Expressing

Journal: Frontiers in Immunology

Article Title: CXCL2 affects macrophage antitumor function and immunotherapy efficacy in esophageal squamous cell carcinoma through calcium signaling

doi: 10.3389/fimmu.2026.1695387

Figure Lengend Snippet: CXCL2 regulated the transition of macrophages to an immune-activated state by mediating cytoplasmic calcium influx. (A) Volcano plot of DEGs between DMSO and CXCL2 treatment groups. (B, C) GO and KEGG analysis of DEGs between DMSO and CXCL2 treatment groups. (D) Flow cytometric analysis of fluo‐3AM positive BMDMs following DMSO and CXCL2 treatment groups. (E) qPCR detecting the indicated genes expression levels on BMDMs in DMSO and CXCL2 treatment groups. (F) Flow cytometry analysis of MHC-II expression on BMDMs in DMSO and CXCL2 treatment groups. (G) qPCR detecting the indicated genes expression levels on BMDMs in the indicated groups. (H) Flow cytometry analysis of MHC-II expression on BMDMs in the indicated groups. * P < 0.05, ** P < 0.01, *** P < 0.001, and NS, not significant; Student’s t-test or one-way ANOVA test. CXCL2, CXC chemokine ligand 2; DMSO, dimethyl sulfoxide; GO, gene ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; MFI, median fluorescence intensity; ANOVA, analysis of variance; BMDMs, bone marrow-derived macrophages; DEG, differentially expressed gene; MHC, major histocompatibility complex; mRNA, messenger RNA; qPCR, quantitative PCR.

Article Snippet: Once tumors became palpable (approximately 5–7 days post-injection), the mice were randomly divided into groups (5 mice per group) and treated as follows: intraperitoneal injection of 100 μg CXCL2 recombinant protein (Cat. HY-P7258; MCE) once every two days, 150 μg anti-PD-1 antibody (Cat. BP0273; BioX Cell) once every three days , or the combination of both.

Techniques: Expressing, Flow Cytometry, Fluorescence, Derivative Assay, Immunopeptidomics, Real-time Polymerase Chain Reaction

Journal: Frontiers in Immunology

Article Title: CXCL2 affects macrophage antitumor function and immunotherapy efficacy in esophageal squamous cell carcinoma through calcium signaling

doi: 10.3389/fimmu.2026.1695387

Figure Lengend Snippet: CXCL2 inhibits tumor growth in the mouse ESCC subcutaneous tumor model. (A) Gross appearance of subcutaneous ESCC tumors in each treatment group. (B) Changes in subcutaneous tumor volumes in each group during the experiment. (C, D) Tumor volumes and weights in each group at the end of the experiment. (E, F) Flow cytometry analysis depicting the proportion of CD11b + F4/80 + MHCII + macrophages and CD3 + CD8 + T cells in each group. * P < 0.05, ** P < 0.01, *** P < 0.001, and NS, not significant; one-way ANOVA test. CXC chemokine ligand 2; ANOVA, analysis of variance; ESCC, esophageal squamous cell carcinoma; MHC, major histocompatibility complex.

Article Snippet: Once tumors became palpable (approximately 5–7 days post-injection), the mice were randomly divided into groups (5 mice per group) and treated as follows: intraperitoneal injection of 100 μg CXCL2 recombinant protein (Cat. HY-P7258; MCE) once every two days, 150 μg anti-PD-1 antibody (Cat. BP0273; BioX Cell) once every three days , or the combination of both.

Techniques: Flow Cytometry, Immunopeptidomics

Journal: Frontiers in Immunology

Article Title: CXCL2 affects macrophage antitumor function and immunotherapy efficacy in esophageal squamous cell carcinoma through calcium signaling

doi: 10.3389/fimmu.2026.1695387

Figure Lengend Snippet: CXCL2 enhances the efficacy of anti-PD-1 antibody in ESCC in vivo . (A) Schematic of the schedule of anti-PD-1 antibody and CXCL2 treatment in AKR cell-derived subcutaneous ESCC mouse models. (B) Changes in subcutaneous tumor volumes in each group during the experiment. (C, D) Tumor volumes and weights in each group at the end of the experiment. (E, F) Flow cytometry analysis depicting the proportion of CD11b + F4/80 + MHCII + macrophages and CD3 + CD8 + T cells in each group. (G) Schematic diagram illustrating the role of CXCL2 in macrophage and the microenvironment immune landscapes of ESCC. Patients with ESCC with immunotherapy responsive typically exhibit a significant infiltration of CXCL2 + macrophages, which facilitate the polarization of macrophages to an immune-activated state through calcium influx, thereby enhancing the cytotoxic function of CD8 + T cells. * P < 0.05, ** P < 0.01, *** P < 0.001, and NS, not significant; one-way ANOVA test. ANOVA, analysis of variance; ESCC, esophageal squamous cell carcinoma; CXC chemokine ligand 2; MHC, major histocompatibility complex; IgG, immunoglobulin G; PD-1, programmed cell death protein-1.

Article Snippet: Once tumors became palpable (approximately 5–7 days post-injection), the mice were randomly divided into groups (5 mice per group) and treated as follows: intraperitoneal injection of 100 μg CXCL2 recombinant protein (Cat. HY-P7258; MCE) once every two days, 150 μg anti-PD-1 antibody (Cat. BP0273; BioX Cell) once every three days , or the combination of both.

Techniques: In Vivo, Derivative Assay, Flow Cytometry, Immunopeptidomics

Journal: Research

Article Title: Gene Signature-Based Drug Screening Reveals Ponatinib Enhances Immunotherapy Efficacy in Triple-Negative Breast Cancer by Reversing MDSC-Mediated Immunosuppressive Tumor Microenvironment

doi: 10.34133/research.0915

Figure Lengend Snippet: High CXCL1 and CXCL2 expression is associated with poor prognosis in patients across multiple cancers. (A) Impact of CXCL1 and CXCL2 expression on RFS in 2,178 patients across 30 cancer types. Log-rank P values were calculated to assess significance. The dashed line indicates P = 0.05. (B) Significance of 9 widely used ICB response biomarkers collected from TIDE in relation to OS across 7 datasets from indicated studies. Red cells: Rho > 0, indicating increased risk. Rho values in the bottom panel represent the average for each biomarker across the 7 datasets (Table ). CXCL1 & CXCL2 , average expression level of CXCL1 and CXCL2 ; MSI, microsatellite instability signature; TIDE, Tumor Immune Dysfunction and Exclusion signature; Merck18, T cell-inflamed signature; TMB, tumor mutation burden. (C) KM plots of OS for patients with breast cancer, grouped by high or low expression of CXCL1 (top) and CXCL2 (bottom). P values were calculated using the log-rank test. Data were derived from RNA-seq and obtained from the KM plotter and TIDE [ , , ]. (D) KM plots of post-progression survival (PPS) for 607 breast cancer patients, grouped by high or low expression of CXCL1 (top) and CXCL2 (bottom). P values were calculated using the log-rank test. Best cutoffs were autocalculated and selected by the KM plotter tool . (E) Genomic alterations of CXCL1 and CXCL2 among all TCGA patients across 30 cancer types. The pie chart shows the percentage of patients with breast cancer and other cancer types. (F) Distribution of the frequency of genomic alterations of CXCL1 and CXCL2 in TCGA cancer patients across 30 cancer types. (G and H) KM plots showing the impact of CXCL1 and CXCL2 genomic alterations on RFS in TCGA patients. Patients were grouped by the presence or absence of CXCL1 and CXCL2 genomic alterations across all TCGA cancer types (G; pan-cancer) or all breast cancer types (H; pan-BRCA). P values were determined using the log-rank test.

Article Snippet: The amount of CXCL1 or CXCL2 protein in the supernatant was measured using a mouse CXCL1- or CXCL2-specific ELISA kit (R&D Systems), according to the manufacturer’s instructions.

Techniques: Expressing, Biomarker Discovery, Mutagenesis, Derivative Assay, RNA Sequencing

Journal: Research

Article Title: Gene Signature-Based Drug Screening Reveals Ponatinib Enhances Immunotherapy Efficacy in Triple-Negative Breast Cancer by Reversing MDSC-Mediated Immunosuppressive Tumor Microenvironment

doi: 10.34133/research.0915

Figure Lengend Snippet: HTS 2 screening identified ponatinib as an antagonist of CXCL1 and CXCL2 expression in diverse cancer cells. (A) Workflow of the drug screening for CXCL1 and CXCL2 using the HTS 2 platform. The compounds were ranked based on their ability to down-regulate the expression of CXCL1 and CXCL2 . The significance of the difference between individual compounds and DMSO was calculated using Student’s t test. −Log 10 ( P value) and log 2 (fold change) were used for compound ranking. Compounds below the dashed line represent the top 10 compounds that effectively decreased the expression of CXCL1 and CXCL2 . (B) Chemical structure of ponatinib (AP24534). (C and D) RT-qPCR analysis of MDA-MB-231 (C) and 4T1 (D) breast cancer cells treated with the indicated concentrations of ponatinib for 24 h. (E) RT-qPCR analysis of MC38 colon cancer cells treated with the indicated concentrations of ponatinib for 24 h. For (C) to (E), values were normalized to the vehicle (DMSO) control group values. Data are presented as means ± SD. (F and G) ELISA analysis of CXCL1 and CXCL2 protein levels in supernatant from 4T1 cells treated with the indicated concentrations of ponatinib (F) or with 1 μM ponatinib at the indicated time points (G). Data are presented as means ± SD. Statistical significance was determined by 2-way ANOVA. *** *P < 0.0001. (H and I) Growth of 4T1 tumors in BALB/c WT mice ( n = 6 mice per group) (H) or MC38 tumors in C57BL/6 WT mice ( n = 10 mice per group) (I) treated with ponatinib or vehicle. Statistical significance was determined by 2-way ANOVA. *** *P < 0.0001. Data are presented as means ± SEM of the indicated number of mice.

Article Snippet: The amount of CXCL1 or CXCL2 protein in the supernatant was measured using a mouse CXCL1- or CXCL2-specific ELISA kit (R&D Systems), according to the manufacturer’s instructions.

Techniques: Expressing, Drug discovery, Quantitative RT-PCR, Control, Enzyme-linked Immunosorbent Assay

Journal: Research

Article Title: Gene Signature-Based Drug Screening Reveals Ponatinib Enhances Immunotherapy Efficacy in Triple-Negative Breast Cancer by Reversing MDSC-Mediated Immunosuppressive Tumor Microenvironment

doi: 10.34133/research.0915

Figure Lengend Snippet: Ponatinib inhibits CXCL1 and CXCL2 expression through the p38α–STAT1 signaling pathway. (A) GO analysis of DEGs between ponatinib-treated versus DMSO-treated MDA-MB-231 (left) and 4T1 (right) cells. DEGs were defined as genes with log 2 (fold change) < −1 and P < 0.05, based on RNA-seq data. P values were determined employing the Fisher’s exact test. (B) Enrichment analysis of the MAPK signaling pathway in MDA-MB-231 (left) and 4T1 (right) cells treated with ponatinib versus DMSO. Data were derived from RNA-seq analysis. NES, normalized enrichment score; P , nominal P value; FDR, false discovery rate. P values were determined using the Kolmogorov–Smirnov (KS) test. (C) Immunoblotting of the indicated proteins in MDA-MB-231 cells treated with 1 μM ponatinib or DMSO. (D) Box plots showing the expression levels of CXCL1 and CXCL2 in cancer cells from breast cancer patients, comparing groups with low expression of MAPK14 to groups with high expression (normalized counts > 0). Data from publicly available scRNA-seq data from 29 breast cancer patients . P values were determined using the Mann–Whitney test. (E) Heatmap of genes involved in the JAK–STAT signaling pathway (top) and STAT1 target genes (bottom) down-regulated by ponatinib in MDA-MB-231 and 4T1 cell lines. (F) Box plots showing the expression levels of CXCL1 or CXCL2 in cancer cells from breast cancer patients, comparing groups with low versus high expression of STAT1 (normalized counts > 0). P values were determined using the Mann–Whitney test. (G) Correlation of CXCL1 or CXCL2 expression with STAT1 expression in TCGA breast cancer patients. Each dot represents an individual patient. Rhos were calculated using TIMER version 2 . P values were determined using the Spearman correlation test. (H) Immunoblotting of the indicated proteins in lysates from MDA-MB-231 cells (left) or 4T1 cells (right) treated with 1 μM ponatinib or vehicle (DMSO). (I) Correlation of the expression levels of MAPK14 (encoding p38α) with STAT1 expression in tumor cells. Each dot represents a breast cancer patient, with the average expression level of all cancer cells from that patient used as the expression level for that individual . P values were determined using the Pearson’s correlation test. (J) Immunoblotting of the indicated proteins in MDA-MB-231 (left) or 4T1 (right) cells with p38α knockdown compared to negative control. (K) Three-dimensional interaction diagram of ponatinib docked in p38α MAPK kinase domain (active site) generated by Schrödinger Maestro, highlighting key binding interactions. (L) In vitro activity of p38α exposed to the indicated concentrations of ponatinib. Data were fitted using a nonlinear regression model (GraphPad Prism) to determine the IC 50 values.

Article Snippet: The amount of CXCL1 or CXCL2 protein in the supernatant was measured using a mouse CXCL1- or CXCL2-specific ELISA kit (R&D Systems), according to the manufacturer’s instructions.

Techniques: Expressing, RNA Sequencing, Derivative Assay, Western Blot, MANN-WHITNEY, Knockdown, Negative Control, Generated, Binding Assay, In Vitro, Activity Assay

Journal: Research

Article Title: Gene Signature-Based Drug Screening Reveals Ponatinib Enhances Immunotherapy Efficacy in Triple-Negative Breast Cancer by Reversing MDSC-Mediated Immunosuppressive Tumor Microenvironment

doi: 10.34133/research.0915

Figure Lengend Snippet: Ponatinib inhibits the infiltration of immunosuppressive MDSCs into TNBC TME by repressing CXCL1 and CXCL2 expression. (A) Growth of 4T1 tumors in BALB/c nude mice treated with ponatinib or vehicle (control, n = 5; ponatinib, n = 8). Tumor volume kinetics were monitored by vernier calipers (left). Statistical significance was determined by 2-way ANOVA. Terminal tumor weight quantification is shown (middle), with statistical significance determined by unpaired 2-tailed Student’s t tests. *** *P < 0.0001. The tumor image (right) shows 4T1 tumors from the indicated groups. (B) Growth of 4T1 tumors in NSG mice treated with ponatinib or vehicle ( n = 6 mice per group). Tumor volume kinetics monitored by vernier calipers (left). Statistical significance was determined by 2-way ANOVA. Terminal tumor weight quantification is shown (middle), with statistical significance determined by unpaired 2-tailed Student’s t tests. ns (not significant), P > 0.05. The tumor image (right) shows 4T1 tumors from the indicated groups. (C) RT-qPCR analysis of Cxcl1 and Cxcl2 mRNA levels in 4T1 tumors from BALB/c WT mice (left) or nude mice (right) receiving ponatinib or control treatments as described in Figs. H and A. Heatmap representing the relative expression of the indicated chemokine genes normalized to Gapdh . Expression scaled from high (red) to low (blue). Each square represents individual tumors from a single mouse (gray squares indicate tumors from mice sacrificed due to the requirement of animal ethics). (D) MDSC identification using publicly available scRNA-seq data from breast cancer patients. Cells were colored by cell types defined in a breast cancer scRNA-seq dataset (left) and by MDSC types predicted using the scPred package (right). MDSCs were identified in a dataset of 29 breast cancer patients receiving ICB therapy (EGAD00001006608) by training on another well-defined breast cancer MDSC dataset ( GSE139125 ) . (E) Percentage of different cell types in the breast cancer TME, grouped by high or low expression of CXCL1 and CXCL2 in scRNA-seq data from breast cancer patients . P values were determined using the chi-square test. (F) Percentage of MDSCs (relative to all myeloid cells) in breast cancer patients with high or low expression of CXCL1 and CXCL2 , calculated using the breast cancer scRNA-seq dataset . (G) Migration of MDSCs toward CM from 4T1 cells treated with ponatinib or DMSO, evaluated using in vitro Transwell migration assays. (H) Migration of MDSCs toward CM from 4T1 cells treated with either DMSO or ponatinib and supplemented with or without recombinant mouse CXCL1 and CXCL2. (I) Transwell migration assays for MDSC migration toward CM from MC38 cells treated with either ponatinib or DMSO. Data are presented as means ± SEM. Statistical significance was assessed using unpaired 2-tailed Student’s t tests. * *P < 0.01, ** *P < 0.001, and *** *P < 0.0001.

Article Snippet: The amount of CXCL1 or CXCL2 protein in the supernatant was measured using a mouse CXCL1- or CXCL2-specific ELISA kit (R&D Systems), according to the manufacturer’s instructions.

Techniques: Expressing, Control, Quantitative RT-PCR, Migration, In Vitro, Recombinant

Journal: Research

Article Title: Gene Signature-Based Drug Screening Reveals Ponatinib Enhances Immunotherapy Efficacy in Triple-Negative Breast Cancer by Reversing MDSC-Mediated Immunosuppressive Tumor Microenvironment

doi: 10.34133/research.0915

Figure Lengend Snippet: Combination treatment with ponatinib enhances the therapeutic efficacy of anti-PD-L1 against TNBC. (A) Expression levels of CXCL1 or CXCL2 and therapeutic responses assessed from publicly available scRNA-seq data for 29 breast cancer patients who received ICB therapy . Cells were colored based on ICB response results (top) and gene expression levels of CXCL1 and CXCL2 (bottom). Es, patients with clonotype expansion ( n = 9); NEs, patients with limited or no clonotype expansion ( n = 20). (B) Receiver operating characteristic (ROC) curves depicting the predictive value of CXCL1 and CXCL2 expression levels for anti-PD-1 therapy response in breast cancer patients. (C) CXCL1 and CXCL2 expression in tumor cells, comparing Es with NEs, calculated using the breast cancer scRNA-seq dataset . P values were determined using the Mann–Whitney test. (D) Expression of known MDSC-related genes [ , , , , ] in MDSCs determined in this study, comparing Es with NEs. MDSCs were identified in the breast cancer scRNA-seq dataset (EGAD00001006608) , through learning from another breast cancer scRNA-seq dataset with MDSC well-defined ( GSE139125 ) , using the cell type prediction method of scPred package . P values were determined using the Mann–Whitney test. (E) Expression of known MDSC-related genes [ , , , , ] in MDSCs, comparing pretreatment with on-treatment conditions. Pre, biopsy collected before anti-PD-1 treatment; On, biopsy collected during subsequent surgery. P values were determined using the Mann–Whitney test. (F) Expression of known MDSC-related genes in the breast TME, comparing Es with NEs from the breast cancer scRNA-seq dataset . P values were determined using the Mann–Whitney test. (G) Expression of known MDSC-related genes in tumor cells, comparing Es with NEs, calculated using the breast cancer scRNA-seq dataset . P values were determined using the Mann–Whitney test. (H) Expression of known MDSC-related genes in MDSCs, comparing across 3 different breast cancer types. P values were determined using the Mann–Whitney test. (I) Expression of known MDSC-related genes in the TME, comparing across 3 different breast cancer types, was calculated using the breast cancer scRNA-seq dataset . P values were determined using the Mann–Whitney test. (J) Schematic diagram of the combination treatment protocol for 4T1-bearing mice; ponatinib (30 mg/kg) or vehicle was administered 4 days per week, and PD-L1 monoclonal antibody (200 μg per injection, twice a week), either alone or in combination, starting when the tumor volume reached 100 mm 3 ( n = 12 to 14 mice per group). (K and L) Growth of 4T1 tumors in BALB/c WT mice treated with the indicated conditions from (J). Tumor volume kinetics were monitored by vernier calipers. Data represent mean ± SEM of n mice. Two-way ANOVA determined statistical significance. * *P < 0.01, ** *P < 0.001, and *** *P < 0.0001. (M) Survival curves for the indicated groups. Statistical significance was determined using the log-rank (Mantel–Cox) test. ns, P > 0.05, * *P < 0.01, and ** *P < 0.001.

Article Snippet: The amount of CXCL1 or CXCL2 protein in the supernatant was measured using a mouse CXCL1- or CXCL2-specific ELISA kit (R&D Systems), according to the manufacturer’s instructions.

Techniques: Drug discovery, Expressing, Gene Expression, MANN-WHITNEY, Injection

Journal: Cell discovery

Article Title: Targeting a chemo-induced adaptive signaling circuit confers therapeutic vulnerabilities in pancreatic cancer.

doi: 10.1038/s41421-024-00720-w

Figure Lengend Snippet: Fig. 3 14-3-3ζ overexpressing PDAC cells increases CXCL2/5 via Yap1 in response to stresses. a Cytokine array analysis of CM from Panc02.shCtrl vs Panc02.shζ cells in 0% FBS culture for 72 h. b Left: WB analysis of CXCL2, CXCL5, and GAPDH (sample processing controls) expression in Panc02.shCtrl and Panc02.shζ cells cultured in 10% FBS or 0% FBS medium for 48 h. Right: WB analysis of CXCL2, CXCL5, and GAPDH (sample processing controls) in 3D cultured PANC-1.shCtrl and PANC-1.shζ cells treated with Gem (20 nM) vs vehicle for 48 h. c WB analysis of CXCL2, CXCL5, 14-3-3ζ, and GAPDH (sample processing controls) in PANC-1.shCtrl and PANC-1.shζ, and 14-3-3ζ-overexpressing PANC-1.shζ cells treated with Gem (20 nM) vs vehicle for 48 h. d WB analyses of Yap1, CXCL2, CXCL5, and GAPDH (sample processing controls) expression in Panc02.shCtrl and Panc02.shYap1 cells in 0% FBS culture for 24 h. Representative data of two independent repeats. e ChIP-qPCR assays of Yap1 binding to CXCL2/5 promoter region in 3D-cultured PANC-1 cells with 24 h of Gem (20 nM) treatment (mean ± SD, t-test, n = 3 biological repeats). f WB analysis of CXCL2, CXCL5, and GAPDH (sample processing controls) in Panc02.shCtrl and Panc02.shNLK sublines cultured in 0.1% FBS for 24 h. Representative data of two independent repeats. g Schematics (left) and relative numbers of proliferating (middle) and apoptotic (right) cells from 3D-cultured PATC53 cells treated with CM from 3D-cultured hPSCs that were activated by adding CM from 8.5 nM Gem-treated 3D-cultured PATC53 cells plus CXCL2 (1 μg/mL) or CXCL5 (3 μg/mL) blocking antibodies for 48 h (mean ± SD, t-test, n = 3 biological repeats). h Schematics and relative cell number of 3D-cultured KPC mT3 cells treated with CM from 3D-cultured mPSCs added with vehicle, recombinant CXCL2 (0.5 ng/mL), or recombinant CXCL5 (0.1 µg/mL) proteins for 48 h (mean ± SD, t-test, n = 3 biological repeats). i Stress-induced 14-3-3ζ-Yap1-CXCL2/5 pathway in PDAC cells activates fibroblasts, which turns on the adaptive response that enables PDAC cells to survive under stress conditions.

Article Snippet: Recombinant mice CXCL2 and CXCL5 were purchased from R&D systems (Minnesota, USA).

Techniques: Expressing, Cell Culture, ChIP-qPCR, Binding Assay, Blocking Assay, Recombinant

Journal: EMBO Reports

Article Title: CMTM3 regulates neutrophil activation and aggravates sepsis through TLR4 signaling

doi: 10.1038/s44319-024-00291-7

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Recombinant Mouse CXCL2 Protein , R&D Systems , 452-M2.

Techniques: Recombinant, Staining, Enzyme-linked Immunosorbent Assay, Red Blood Cell Lysis, cDNA Synthesis, SYBR Green Assay, Real-time Polymerase Chain Reaction, Isolation, Membrane, Software