human mouse recombinant activin a  (R&D Systems)

Journal: Frontiers in Immunology

Article Title: IGLC3 - tumor cells drive chemoresistance in colorectal cancer by polarizing SPP1 + macrophages via the CD44-Wnt-BTF3 axis

doi: 10.3389/fimmu.2026.1731216

Figure Lengend Snippet: IGLC3 - tumor cells promote SPP1 + macrophage polarization. (A) TSNE of 5 macrophage cell clusters in scRNA-seq of colorectal tumor tissues, including C1QC&LYVE1 + macrophages, C1QC + macrophages, C1QC&ISG15 + macrophages, ISG15 + macrophages and SPP1 + macrophages. (B) Heatmap of top differential gene expression between each macrophage subtypes. (C) Dot plot of inflammatory macrophages marker genes in each macrophage subtype. (D) The developmental trajectory between macrophage subtypes by monocle2 analysis. (E) Schematic diagram of macrophage and tumor cells co-culture model. (F) Heatmap of C1QC, LYVE1, SPP1 and ISG15 at mRNA level in THP-1 derived M0 macrophages co-cultured with vector and IGLC3 overexpressed HCT116/SW480 cells. The PBS treated THP-1 cells were set as control. (G) mRNA expression of CD206, IL-10 and Arg1 in patients derived SPP1 + macrophages (tumor tissues), and peripheral blood mononuclear cells derived M0 and M2 macrophages (blood). (H) Immunostaining of CD68 and SPP1 in patients derived colorectal tumor tissues. The CD68 and SPP1 + cells distribution was compared between patients with stage I~II (low stage) or stage III~IV (high stage), with metastasis or not, and responding to chemotherapy or not. (I) Volcano plot of differentially expressed cytokine-related genes between IGLC3 + and IGLC3 - tumor cells identified from our scRNA-seq dataset ( GSE166555 ). (J, K) Elisa of TGF-β and CXCL3 in supernatant of vector and IGLC3 overexpressed HCT116/SW480 cells. (L) mRNA expression of SPP1, ISG15, CD80 and CD68 in THP-1 derived M0 macrophages treated with TGF-β (10 ng/ml) and CXCL3 (50 ng/ml). All experiments were independently repeated at least three times with consistent results. Data are presented as mean ± SD, and statistical analyses were performed using t test. *p<0.05, **p<0.01.

Article Snippet: The following reagents were used: recombinant human TGF-β, CXCL3, fibronectin (FN), SPP1, 5-fluorouracil (5-Fu), and oxaliplatin (Oxa) (all from R&D Systems, USA).

Techniques: Gene Expression, Marker, Co-Culture Assay, Derivative Assay, Cell Culture, Plasmid Preparation, Control, Expressing, Immunostaining, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Oncogenic KRAS cells use Wnt signalling and cell dormancy to override homeostatic cell elimination mechanisms in adult pancreas

doi: 10.1101/2024.02.13.579930

Figure Lengend Snippet: Heatmaps of A Wnt signalling pathway or B Wnt/β-catenin pathway related genes (rows) in Control and KrasG12D (KC) cell transcriptomes (columns). The heatmaps show row z-scores for each gene obtained from the RNA sequencing experiment. Data from three samples (three mice pooled per sample) per genotype are shown. Only genes with z-scores of a foldchange of 2 are shown in the Wnt pathway panel. The full list of genes can be found in supplementary table S11. C Wnt5a mRNA reads relative to control reads in the three samples in Control and three samples in KrasG12D (KC) obtained from the RNA sequencing experiment. Graph represents mean +/- s.d. reads. D Confocal images of pancreas tissues harvested at 35-days p.i. from Control, KrasG12D (KC) mice. Tissues were stained with anti- RFP (red) and anti-β-catenin (cyan) antibodies and Hoechst (blue). Dashed boxes represent digital zoom images of representative cell-cell contacts. Scale bar, 50µm. E Mean fluorescence intensity of β-catenin at the boundary between RFP positive and negative cells in Control and KrasG12D (KC) tissues harvested at 35 days p.i. Mean fluorescence intensity is relative to the background. Fluorescence was measured in ROIs, separated by a constant distance, along the cell-cell boundary. SuperPlot shows all the quantified ROIs (circles) and the mean for each mouse (n=3 samples; triangle, square and diamond). The graph shows mean and +/- s.d. for the three mice. Student’s t test was used to analyse the data. *p=0.0347. F Mean fluorescence intensity of β-catenin in the nuclei of RFP positive cells in Control and KrasG12D (KC) tissues harvested at 35 days p.i. Mean fluorescence intensity is relative to the background. SuperPlot shows all the quantified nuclei (circles) and the mean for each mouse (n=3 samples; triangle, square and diamond). The graph shows mean and +/- s.d. for the three mice. Student’s t test was used to analyse the data. ns=0.3035. G Confocal images of pancreas tissues harvested at 35-days p.i. from Control, KrasG12D (KC) and KrasG12D high dose tamoxifen (KC megadose) mice and stained with anti-RFP (red) and anti-CD44 (cyan) antibodies and Hoechst (blue). Scale bar, 50µm. H Mean fluorescence intensity of CD44 at the boundary between RFP positive and RFP negative cells in Control, KrasG12D low dose tamoxifen (KC) and KrasG12D high dose tamoxifen (KC Megadose) tissues harvested at 35 days p.i. CD44 fluorescence was quantified and reported as in F. SuperPlot shows all the quantified ROIs (circles) and the mean for each mouse (n=3 samples; triangle, square and diamond). The graph shows mean and +/- s.d. for the three mice. Student’s t test was used to analyse the data. **p=0.0047; ***p=0.0009; ns=0.2136. I Mean fluorescence intensity of CD44 in entire RFP positive clusters. Mean fluorescence intensity is relative to the background. SuperPlot shows all the quantified clusters (circles) and the mean for each mouse (n=3 samples; triangle, square and diamond). The graph shows mean and +/- s.d. for the three mice. Student’s t test was used to analyse the data. *p<0.05; ns=0.3916.

Article Snippet: For Wnt5a experiments tetracycline induced GFP-RasV12 cells were treated with 100ng/mL recombinant human/mouse Wnt5a (R&D Systems) or PBS (control-Sigma-Aldrich) for 24h and then mixed with parental MDCK cells.

Techniques: Control, RNA Sequencing, Staining, Fluorescence

Journal: bioRxiv

Article Title: Oncogenic KRAS cells use Wnt signalling and cell dormancy to override homeostatic cell elimination mechanisms in adult pancreas

doi: 10.1101/2024.02.13.579930

Figure Lengend Snippet: A Confocal images of extruded and non-extruded siScr-GFP-RasV12 and siMyc 1+2-GFP-RasV12 cells for 48h with DMSO or WNT-974. Fixed cells were stained with Hoechst (blue). Scale bar, 100 µm. B The proportion of non-extruded GFP-RasV12 cells transiently expressing siRNA Myc oligos (siMyc1+2) relative to non-extruded GFP-RasV12 cells expressing siRNA scrambled (siScr). Cells were treated with DMSO (white bars) or WNT-974 porcupine inhibitor (dashed bars). The data represent mean +/- s.d. for at least 300 cells from three independent repeats. Student’s t test was used to analyse the data. ***p<0.0005. C Confocal images of extruded and non-extruded GFP-RasV12 cells pre-treated with PBS/Wnt5a and treated for 48h with DMSO/WNT-974/OMP-18R5. OMP-18R5 is a Frizzled receptor antagonist. Fixed cells were stained with Hoechst (blue). Scale bar, 100 µm. D, E. GFP-RasV12 non-extruded cells pre-treated with PBS or Wnt5a recombinant protein 24h before mixing with MDCK cells (1:50). Six hours after mixing and plating, cells were treated with D DMSO or WNT-974 porcupine inhibitor or E OMP-18R5. The data represent mean +/- s.d. for at least 300 cells from three independent repeats. Student’s t test was used to analyse the data, ***p<0.0005, **p<0.005, *p<0.05. F Experimental design for Wnt inhibition experiments in vivo . 6–8-week-old KC ( Kras G12D/+ ) mice were induced with low-dose tamoxifen. Thirty-five days later, mice were treated with the porcupine inhibitor WNT-974 or vehicle for 28 days. Illustration created with BioRender.com. G Representative images of endogenous RFP fluorescence in KC pancreas tissue sections harvested 28-days post treatment with vehicle or WNT-974. Scale bar, 500µm. H RFP fluorescence per tissue area in KC mice treated with WNT-974 or vehicle and relative to vehicle treated tissues. Data represent mean per mouse (n=4-6 samples) and +/- s.d. Student’s t test was used to analyse the data, ***p=0.0003.

Article Snippet: For Wnt5a experiments tetracycline induced GFP-RasV12 cells were treated with 100ng/mL recombinant human/mouse Wnt5a (R&D Systems) or PBS (control-Sigma-Aldrich) for 24h and then mixed with parental MDCK cells.

Techniques: Staining, Expressing, Recombinant, Inhibition, In Vivo, Fluorescence

Journal: Oncoimmunology

Article Title: Blockade of TGF-β signaling with novel synthetic antibodies limits immune exclusion and improves chemotherapy response in metastatic ovarian cancer models

doi: 10.1080/2162402X.2018.1539613

Figure Lengend Snippet: Affinities of lead inhibitory antibodies for human and mouse TGFBR2.

Article Snippet: The mouse TGFBR2-Fc (catalog number 532-R2) and human TGFBR1-Fc (catalog number 3025-BR) were purchased from R&D Systems.

Techniques: Clone Assay

Journal: Oncoimmunology

Article Title: Blockade of TGF-β signaling with novel synthetic antibodies limits immune exclusion and improves chemotherapy response in metastatic ovarian cancer models

doi: 10.1080/2162402X.2018.1539613

Figure Lengend Snippet: Genes upregulated in SKOV3 tumors treated with anti-TGFBR2 IgG-8311.

Article Snippet: The mouse TGFBR2-Fc (catalog number 532-R2) and human TGFBR1-Fc (catalog number 3025-BR) were purchased from R&D Systems.

Techniques:

Journal: Oncoimmunology

Article Title: Blockade of TGF-β signaling with novel synthetic antibodies limits immune exclusion and improves chemotherapy response in metastatic ovarian cancer models

doi: 10.1080/2162402X.2018.1539613

Figure Lengend Snippet: Genes downregulated in SKOV3 tumors treated with anti-TGFBR2 IgG-8311.

Article Snippet: The mouse TGFBR2-Fc (catalog number 532-R2) and human TGFBR1-Fc (catalog number 3025-BR) were purchased from R&D Systems.

Techniques: