mouse anti cxcl5  (R&D Systems)

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: a Cytokine array analysis of CM from Panc02.shCtrl vs Panc02.sh ζ 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.sh ζ 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.sh ζ 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.sh ζ 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.sh Yap1 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.sh NLK 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: Antibody to mouse CXCL5 (bs-2549R) was from Bioss antibodies (Massachusetts, USA).

Techniques: Expressing, Cell Culture, Binding Assay, Blocking Assay, Recombinant

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: a WB analyses of Cox2 and GAPDH (sample processing controls) in mPSCs (3D culture) incubated with recombinant CXCL2 (0.5 ng/mL) or CXCL5 (0.1 µg/mL) for 24 h. Representative data of two independent repeats. b WB analysis of Cox2 and GAPDH (sample processing controls) in NIH3T3 cells treated with CM from Panc02.shCtrl, Panc02.sh CXCL2 , or Panc02.sh CXCL5 cells cultured in 0% FBS for 72 h. Representative data of two independent repeats. c PGE2 concentration in CM from NIH3T3 cells treated with recombinant CXCL2 (0.5 ng/mL) or CXCL5 (0.1 µg/mL) for 48 h (mean ± SD, t -test, n = 3 biological repeats). d Relative numbers of Panc02 cells treated with PGE2 in 0% FBS for 48 h (mean ± SD, t -test, n = 3 biological repeats). e Relative numbers of PANC-1 cells in upper chambers of Transwell units 3D-cocultured with hPSCs in lower chambers of Transwell units with or without Cel (4 nM) and Gem (20 nM) for 72 h (mean ± SD, t -test, n = 3 biological repeats). f Relative number of Panc02 cells treated with CM from WT mPSCs or Cox2 –/– mPSCs activated by CM from Panc02 cells in 0% FBS for 48 h (mean ± SD, t -test, n = 3 biological repeats). g RPPA analysis of NIH3T3 cells treated with CM collected from Panc02.shCtrl and Panc02.sh ζ cells cultured in 10% or 0% FBS medium. h WB analysis of pT346-NDRG1, NDRG1, and GAPDH (sample processing controls) in 3D-cultured hPSCs treated for 24 h with CM collected from 3D-cultured PANC-1.shCtrl and PANC-1.sh ζ cells treated with vehicle/Gem (20 nM) for 24 h. Representative data of two independent repeats. i WB analysis of pT346-NDRG1, NDRG1, pS473-Akt, Akt, and GAPDH (sample processing controls) expression in 3D-cultured hPSCs treated with recombinant CXCL2 (15 ng/mL) or CXCL5 (25 ng/mL) for 24 h. Representative data of two independent repeats. j WB analysis of Rictor, pT346-NDRG1, NDRG1, Cox2, and GAPDH (sample processing controls) protein in NIH3T3.shCtrl and NIH3T3.sh Rictor cells treated for 24 h with recombinant CXCL2 (0.5 ng/mL) or recombinant CXCL5 (0.1 µg/mL). Representative data of two independent repeats. k, l IHC staining of indicated proteins of PDAC tumor tissues from KPC and KPC -ζ fl/fl mice with indicated treatments (mean ± SD, t -test, n = 5 biological repeats, scale bar: 20 µm). m PDAC cells and fibroblasts together create the adaptive stress response circuit, which is essential for PDAC cell adaptation to stresses, including nutrient deprivation and chemotherapy-induced genotoxicity. Stressors promote the Yap1-CXCL2/5 signaling axis via NLK in 14-3-3ζ-overexpressing PDAC cells, leading to the activation of the CXCR2-mTORC2-Cox2-PGE2 pathway in fibroblasts. Reciprocally, PGE2 from fibroblasts promotes PDAC cell survival and adaptive resistance to Gem.

Article Snippet: Antibody to mouse CXCL5 (bs-2549R) was from Bioss antibodies (Massachusetts, USA).

Techniques: Incubation, Recombinant, Cell Culture, Concentration Assay, Expressing, Immunohistochemistry, Activation Assay

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: The blocking antibodies to mouse CXCL2 (MAB452), mouse CXCL5 (MAB433), human CXCL5 (MAB254), and control antibody (MAB0061) were purchased from R&D Systems and human CXCL2 (311001) was purchased from Biolegend (California, USA).

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

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Tumor Microenvironment Responsive CD8 + T Cells and Myeloid-Derived Suppressor Cells to Trigger CD73 Inhibitor AB680-Based Synergistic Therapy for Pancreatic Cancer.

doi: 10.1002/advs.202302498

Figure Lengend Snippet: Figure 6. AB680 regulates CXCL5 released by tumor cells and promotes MDSC accumulation. A) Heatmap of immune-related differential genes in the RNA-seq data of control and AB680-treated tumors (n = 3 samples per group). B,C) Over-Representation Analysis B) and Gene Set Enrichment Analysis (C) of the above RNA-seq data. D) Heatmap of 23 different mouse cytokine expression levels in serums obtained at the time of tumor collection, as shown in Figure 3E using Bio-Plex Pro Mouse Cytokine 23-plex immunoassay (n = 4 samples per group). E) The protein level of CXCL5 in mouse serums from the indicated treatment groups. Data presented as mean ± SD, **p < 0.01 in Student’s t-test. F) Dot plot of Cxcl5 expression in the identified populations

Article Snippet: Formalin-fixed, paraffin-embedded sections of humans and mice were processed and incubated with primary antibodies: CD73 (Proteintech, 12231-1-AP), CK19 (Proteintech, 10712-1-AP), α-SMA (Boster, BM0002), CD8 (Abcam, ab209775), granzyme B (Abcam, ab4059), CXCR2 (Proteintech, 19538-1- AP), CXCL5 (R&D Systems, AF433), LY6G (Servicebio, GB11229), Ki-67 (CST, 9129), and cleaved caspase 3 (CST, 9664), followed by biotinylated or fluorescent secondary antibodies.

Techniques: RNA Sequencing, Control, Expressing