anti dkk3 primary antibody  (Proteintech)

Journal: Molecular Medicine Reports

Article Title: Dickkopf-3 upregulation mediates the cardioprotective effects of curcumin on chronic heart failure

doi: 10.3892/mmr.2018.8783

Figure Lengend Snippet: Effects of curcumin on DKK-3, p38, JNK and ASK1 protein expression levels. (A) Representative images of DKK-3 and (B) quantification of its expression levels in LVAW, as evaluated by immunohistochemical staining. (C) Representative images of DKK-3 and (D) quantification of its expression, as evaluated by western blot analysis. (E) Representative images of p-p38, p-JNK and p-ASK1 and quantification of (F) p-p38; (G) p-JNK and (H) p-ASK1 expression levels, as measured by western blot analysis. Magnification, ×200. Scale bar=50 µm. All data are expressed as the mean ± stadard deviation, n=10. *P<0.05. ASK1, apoptosis signal-regulating kinase 1; CHF, chronic heart failure; Con, control; Cur, curcumin; DKK-3, Dickkopf-related protein 3; LVAW, left ventricular posterior wall thickness; p, phosphorylated; T, total; p38, p38 mitogen-activated protein kinase; JNK, c-Jun N-terminal kinase.

Article Snippet: Primary rabbit polyclonal anti-DKK-3 antibody (1:200; cat. on. bs-2686R; BIOSS) and secondary antibody mouse anti-rabbit IgM/HRP (1:200, bs-0369M-HRP, BIOSS) were used in the staining.

Techniques: Expressing, Immunohistochemical staining, Staining, Western Blot

Journal: Scientific Reports

Article Title: Folic acid inhibits the Wnt/β-catenin pathway by upregulating DKK3 to exert anti-tumor effects in cervical squamous cell carcinoma

doi: 10.1038/s41598-025-32762-9

Figure Lengend Snippet: Establishment of DKK3-manipulated SiHa cell models with altered Wnt/β-catenin signaling activity. ( a , f ) Brightfield and fluorescence microscopy images after transfection with different lentivirus in SiHa cells cultured in medium (scale bar: 100 μm). ( b , g ) DKK3 mRNA expression in different treated cells ( n = 3). ( c - e , h - j ) Western blotting and corresponding quantitative expression analysis of DKK3 and β-catenin ( n = 3). Original blots are presented in Supplementary Fig. . * P < 0.05, ** P < 0.01, and *** P < 0.001 represent significant difference.

Article Snippet: Next, anti-DKK3 primary antibody (1:100 dilution; cat # 66758-1-lg, Proteintech, Wuhan, China) was applied to the sections for overnight incubation at 4 °C after a 30-min blocking step.

Techniques: Activity Assay, Fluorescence, Microscopy, Transfection, Cell Culture, Expressing, Western Blot

Journal: Scientific Reports

Article Title: Folic acid inhibits the Wnt/β-catenin pathway by upregulating DKK3 to exert anti-tumor effects in cervical squamous cell carcinoma

doi: 10.1038/s41598-025-32762-9

Figure Lengend Snippet: Cell proliferation, migration and invasion are affected by DKK3 expression in vitro. ( a , i ) CCK-8 assay detected the ability of cell proliferation ( n = 3). ( b - c , j - k ) Colony formation assay and count of different groups of SiHa cells( n = 3). ( d - e , l - m ) Representative wound healing assay images and analysis of different groups of SiHa cells ( n = 3). ( f - h , n - p ) Representative cell migration and invasion images and analysis of SiHa cells from different groups using transwell assay ( n = 3). * P < 0.05, ** P < 0.01, and *** P < 0.001 represent significant difference.

Article Snippet: Next, anti-DKK3 primary antibody (1:100 dilution; cat # 66758-1-lg, Proteintech, Wuhan, China) was applied to the sections for overnight incubation at 4 °C after a 30-min blocking step.

Techniques: Migration, Expressing, In Vitro, CCK-8 Assay, Colony Assay, Wound Healing Assay, Transwell Assay

Journal: Scientific Reports

Article Title: Folic acid inhibits the Wnt/β-catenin pathway by upregulating DKK3 to exert anti-tumor effects in cervical squamous cell carcinoma

doi: 10.1038/s41598-025-32762-9

Figure Lengend Snippet: FA inhibits cell viability, migration and invasion by upregulating DKK3 and reducing β-catenin protein abundance. ( a ) schematic illustration of the DKK3 regulation after cells treated with FA. ( b ) Cell viability of SiHa cells treated with different concentrations of FA ( n = 3). ( c )Cell viability of SiHa cells treated with different time course of FA ( n = 3). ( d ) Flow cytometry analysis for cell apoptosis of SiHa cells with different treatments ( n = 3). ( e ) Representative images for flow cytometry of SiHa cells with different treatments. ( f - g ) Representative wound healing assay images and analysis of FA treatment in SiHa cells ( n = 3). ( h ) Representative images of cell migration and invasion in SiHa cells treated with FA, using transwell assay. ( i - j ) Corresponding quantitative analysis of migration and invasion ( n = 3). (k) DKK3 mRNA levels in SiHa cells treated with different concentrations of FA ( n = 3). ( l – n ) Protein expression analysis of DKK3 and β‑catenin in SiHa cells under different FA treatments: representative western blot images (l) and corresponding quantification of DKK3 (m) and β‑catenin (n) ( n = 3). ( o ) Rescue experiments: DKK3 mRNA levels in DKK3‑knockdown SiHa cells treated with different concentrations of FA ( n = 3). ( p – r ) Rescue experiments: Protein expression analysis in DKK3‑knockdown SiHa cells under FA treatment: representative western blot images ( p ) and corresponding quantification of DKK3 ( q ) and β‑catenin ( r ) ( n = 3). Original blots are presented in Supplementary Fig. .* P < 0.05, ** P < 0.01, and *** P < 0.001 represent significant difference.

Article Snippet: Next, anti-DKK3 primary antibody (1:100 dilution; cat # 66758-1-lg, Proteintech, Wuhan, China) was applied to the sections for overnight incubation at 4 °C after a 30-min blocking step.

Techniques: Migration, Quantitative Proteomics, Flow Cytometry, Wound Healing Assay, Transwell Assay, Expressing, Western Blot

Journal: Cancers

Article Title: DKK3, Downregulated in Invasive Epithelial Ovarian Cancer, Is Associated with Chemoresistance and Enhanced Paclitaxel Susceptibility via Inhibition of the β-Catenin-P-Glycoprotein Signaling Pathway

doi: 10.3390/cancers14040924

Figure Lengend Snippet: Immunoreactivity of DKK3 and survival curves for patients with invasive serous ovarian cancer. ( a ) DKK3 immunoreactivity in normal epithelium and serous tumors. ( b ) Immunoreactivity of each score in invasive serous carcinoma. ( c ) Kaplan–Meier curves for disease-free survival. Magnification 200×.

Article Snippet: Nonreactive blocking was performed with 1.0% horse serum in Tris-buffered saline (TBS), pH 6.0, for 3 min. Primary goat polyclonal antibody against human DKK3 (R&D Systems, Minneapolis, MN, USA), diluted 1:100 in phosphate-buffered saline (PBS) (pH 7.4), was applied and incubated for 1 h at 37 °C in a humidified chamber.

Techniques:

Journal: Cancers

Article Title: DKK3, Downregulated in Invasive Epithelial Ovarian Cancer, Is Associated with Chemoresistance and Enhanced Paclitaxel Susceptibility via Inhibition of the β-Catenin-P-Glycoprotein Signaling Pathway

doi: 10.3390/cancers14040924

Figure Lengend Snippet: Expression of DKK3 in different ovarian tissue samples.

Article Snippet: Nonreactive blocking was performed with 1.0% horse serum in Tris-buffered saline (TBS), pH 6.0, for 3 min. Primary goat polyclonal antibody against human DKK3 (R&D Systems, Minneapolis, MN, USA), diluted 1:100 in phosphate-buffered saline (PBS) (pH 7.4), was applied and incubated for 1 h at 37 °C in a humidified chamber.

Techniques: Expressing

Journal: Cancers

Article Title: DKK3, Downregulated in Invasive Epithelial Ovarian Cancer, Is Associated with Chemoresistance and Enhanced Paclitaxel Susceptibility via Inhibition of the β-Catenin-P-Glycoprotein Signaling Pathway

doi: 10.3390/cancers14040924

Figure Lengend Snippet: Clinicopathological parameters and disease-free survival analysis of prognostic factors in 42 patients with serous adenocarcinoma.

Article Snippet: Nonreactive blocking was performed with 1.0% horse serum in Tris-buffered saline (TBS), pH 6.0, for 3 min. Primary goat polyclonal antibody against human DKK3 (R&D Systems, Minneapolis, MN, USA), diluted 1:100 in phosphate-buffered saline (PBS) (pH 7.4), was applied and incubated for 1 h at 37 °C in a humidified chamber.

Techniques: Expressing

Journal: Cancers

Article Title: DKK3, Downregulated in Invasive Epithelial Ovarian Cancer, Is Associated with Chemoresistance and Enhanced Paclitaxel Susceptibility via Inhibition of the β-Catenin-P-Glycoprotein Signaling Pathway

doi: 10.3390/cancers14040924

Figure Lengend Snippet: Clinicopathological characteristics of women with/without DKK3 expression.

Article Snippet: Nonreactive blocking was performed with 1.0% horse serum in Tris-buffered saline (TBS), pH 6.0, for 3 min. Primary goat polyclonal antibody against human DKK3 (R&D Systems, Minneapolis, MN, USA), diluted 1:100 in phosphate-buffered saline (PBS) (pH 7.4), was applied and incubated for 1 h at 37 °C in a humidified chamber.

Techniques: Expressing

Journal: Cancers

Article Title: DKK3, Downregulated in Invasive Epithelial Ovarian Cancer, Is Associated with Chemoresistance and Enhanced Paclitaxel Susceptibility via Inhibition of the β-Catenin-P-Glycoprotein Signaling Pathway

doi: 10.3390/cancers14040924

Figure Lengend Snippet: Characteristics of paclitaxel-resistant cells. ( a , b ) Cells were seeded in triplicates in 96-well plates in 0.1 mL culture medium at a density of 1 × 10 4 cells/well. After 24 h, the cells were treated with paclitaxel. MTT assay performed 48 h after treatment showed that the paclitaxel-resistant cells (TOV-21G/PTX and OV-90/PTX) had higher IC 50 than the parental cells. ( c , d ) Paclitaxel treatment reduced the viability of parental cells, while the paclitaxel-resistant cells continued to grow. ( e ) Western blot analysis revealed that DKK3 was lost and that β-catenin and P-glycoprotein were upregulated in paclitaxel-resistant cells (Res) compared to that in their parent cells (Con). β-Actin was used as the loading control. * p < 0.01.

Article Snippet: Nonreactive blocking was performed with 1.0% horse serum in Tris-buffered saline (TBS), pH 6.0, for 3 min. Primary goat polyclonal antibody against human DKK3 (R&D Systems, Minneapolis, MN, USA), diluted 1:100 in phosphate-buffered saline (PBS) (pH 7.4), was applied and incubated for 1 h at 37 °C in a humidified chamber.

Techniques: MTT Assay, Western Blot, Control

Journal: Cancers

Article Title: DKK3, Downregulated in Invasive Epithelial Ovarian Cancer, Is Associated with Chemoresistance and Enhanced Paclitaxel Susceptibility via Inhibition of the β-Catenin-P-Glycoprotein Signaling Pathway

doi: 10.3390/cancers14040924

Figure Lengend Snippet: Anti-proliferative effect of the secreted DKK3 on paclitaxel-resistant cells. ( a ) Western blotting results show DKK3 protein levels in the control and DKK3 conditioned medium (CM). MTT assay performed after 24 h of incubation with CM (*** p < 0.001). ( b ) The viability of TOV-21G and TOV-21G/PTX cells after treatment with DKK3 CM diluted to 0%, 50%, and 90%, respectively, with control CM (** p < 0.01). ( c ) The OV-90 and OV-90/PTX cells were incubated with CM with or without DKK3. Western blotting results show DKK3 protein levels in the control and DKK3 CM. MTT assay after 24 h of treatment (*** p < 0.001). ( d ) Western blotting reveals recombinant human DKK3 levels. Two concentrations of the protein (10 μg/mL and 50 μg/mL) were used to treat cells for 72 h (* p < 0.05). ( e , f ) TOV-21G/PTX and OV-90/PTX cells were treated with CM with or without paclitaxel (100 ng/mL and 200 ng/mL, respectively) (** p < 0.01). ( g ) 3D spheroids of OV-90 and OV-90/PTX cells were generated in a microwell array and the images were captured after incubation for 6 d (magnification, 40×; scale bar 300 μm). ( h ) Spheroid viabilities and diameters were compared. The asterisks on the graph denote statistically significant differences ( p < 0.01) between the DKK3 CM group and the control CM group. The diameter of spheroids in the DKK3 CM group was compared to that in the DKK3 CM with paclitaxel group (#, p < 0.01). ( i , j ) OV-90/PTX and TOV-21G/PTX cells were treated as mentioned above and migration rates were evaluated using the migration assay. ( k , l ) The paclitaxel-resistant cells were incubated with control and DKK3 CMs. At the indicated time points, the cells were harvested and subjected to Western blotting. The graphs were plotted based on the band densities measured using the ImageJ software. * p < 0.05.

Article Snippet: Nonreactive blocking was performed with 1.0% horse serum in Tris-buffered saline (TBS), pH 6.0, for 3 min. Primary goat polyclonal antibody against human DKK3 (R&D Systems, Minneapolis, MN, USA), diluted 1:100 in phosphate-buffered saline (PBS) (pH 7.4), was applied and incubated for 1 h at 37 °C in a humidified chamber.

Techniques: Western Blot, Control, MTT Assay, Incubation, Recombinant, Generated, Migration, Software

Journal: Cancers

Article Title: DKK3, Downregulated in Invasive Epithelial Ovarian Cancer, Is Associated with Chemoresistance and Enhanced Paclitaxel Susceptibility via Inhibition of the β-Catenin-P-Glycoprotein Signaling Pathway

doi: 10.3390/cancers14040924

Figure Lengend Snippet: Secreted DKK3 enhanced paclitaxel susceptibility via inhibition of the β-catenin-P-glycoprotein signaling pathway. ( a , b ) The cells were incubated with control and DKK3 CM. Immunofluorescence analysis after 24 h showed that FLAG-DKK3 was present in the perinuclear area, attenuating TR-non-phospho-β-catenin signaling. Scale bar, 50 μm. ( c , d ) The cells were incubated with control and DKK3 CM and subjected to Western blotting. The graphs were plotted based on the band densities (* p < 0.01). ( e , f ) To activate endogenous β-catenin, cells were treated with LiCl for 24 h and subjected to Western blot analysis. β-Actin was used as the loading control. The intensities of the Western blot bands were normalized to that of β-actin for comparison (* p < 0.01).

Article Snippet: Nonreactive blocking was performed with 1.0% horse serum in Tris-buffered saline (TBS), pH 6.0, for 3 min. Primary goat polyclonal antibody against human DKK3 (R&D Systems, Minneapolis, MN, USA), diluted 1:100 in phosphate-buffered saline (PBS) (pH 7.4), was applied and incubated for 1 h at 37 °C in a humidified chamber.

Techniques: Inhibition, Incubation, Control, Immunofluorescence, Western Blot, Comparison

Journal: Science translational medicine

Article Title: Suppression of stromal-derived Dickkopf-3 (DKK3) inhibits tumor progression and prolongs survival in pancreatic ductal adenocarcinoma

doi: 10.1126/scitranslmed.aat3487

Figure Lengend Snippet: Dkk3 expression was measured in HPSCs and PDAC cell lines by RTPCR (A) and qPCR (B) in monoculture and coculture. Striped bars indicate expression in HPSCs after coculture with PDAC cells. (C) Dkk3 expression in human PDAC and normal pancreatic tissue was determined by Affymetrix array. (D) IHC of DKK3 in a tissue microarray of human PDAC. Shown are representative fields from PDAC expressing low and high amounts of DKK3 with normal pancreas and negative controls. (E) DKK3 concentrations were measured by enzyme-linked immunosorbent assay (ELISA) in plasma samples from patients with PDAC, CP, or no pancreatic disease and in conditioned medium (CM) from HPSCs (HPSC-CM). (F) In a GEMM of PDAC, DKK3 is expressed early in development with CP and PanIN lesions and increases in PDAC. Scale bars, 200 μm. (G) Relative expression of Dkk3 in the GEMM of PDAC and in cancer cells isolated from GEMM tumors was quantified by Affymetrix. *P < 0.05, ***P < 0.001. Data are means ± SEM.

Article Snippet: Primary rabbit anti-mouse DKK3 Ab was obtained from Proteintech Inc., goat anti-human DKK3 was obtained from Abcam, and rabbit anti-goat secondary Ab was obtained from Jackson ImmunoResearch Laboratories. mAbs against mouse α-SMA, Ki67, CD3, and CD8 were purchased from Abcam, Thermo Fisher Scientific, Santa Cruz Biotechnology, and BioLegend, respectively.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Microarray, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Isolation

Journal: Science translational medicine

Article Title: Suppression of stromal-derived Dickkopf-3 (DKK3) inhibits tumor progression and prolongs survival in pancreatic ductal adenocarcinoma

doi: 10.1126/scitranslmed.aat3487

Figure Lengend Snippet: (A) HPSC proliferation was measured by MTT [3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide] after treatment with PBS or rhDKK3 (10 μg/ml). OD490, optical density at 490 nm. DKK3 was silenced in HPSCs by shDKK3 (fig. S1B), cell proliferation was measured by MTT assay (B), and migration was determined at 24 hours (C). Control cells were transfected with scrambled short hairpin RNA (shRNA). (D) Panc1 cells were treated with rhDKK3 (10 μg/ml) or serum-free medium control, and cell migration and invasion were measured after 24 hours. (E) Panc1 cells were stably silenced for DKK3, and (E) cell proliferation and (F) colony formation in soft agar were measured. (G) BxPC3 cell migration was measured after treatment with CM from HPSCs or HPSCs silenced for DKK3. HPF, high-power field. (H) Soft agar colony formation in gemcitabine and (I) apoptosis were determined in chemosensitive L3.6pl cells expressing DKK3 compared with transfection controls. (J) Gemcitabine-induced apoptosis was measured in chemoresistant HS766T cells silenced for DKK3. *P < 0.01, **P < 0.001, ***P < 0.0001, ****P < 0.00001. Scale bars, 200 μm.

Article Snippet: Primary rabbit anti-mouse DKK3 Ab was obtained from Proteintech Inc., goat anti-human DKK3 was obtained from Abcam, and rabbit anti-goat secondary Ab was obtained from Jackson ImmunoResearch Laboratories. mAbs against mouse α-SMA, Ki67, CD3, and CD8 were purchased from Abcam, Thermo Fisher Scientific, Santa Cruz Biotechnology, and BioLegend, respectively.

Techniques: MTT Assay, Migration, Control, Transfection, shRNA, Stable Transfection, Expressing

Journal: Science translational medicine

Article Title: Suppression of stromal-derived Dickkopf-3 (DKK3) inhibits tumor progression and prolongs survival in pancreatic ductal adenocarcinoma

doi: 10.1126/scitranslmed.aat3487

Figure Lengend Snippet: (A) Phosphorylation of p65 and IκBα induced by DKK3 treatment (10 μg/ml) was determined by Western blotting. Relative protein loading was shown by using anti–β-actin Ab. (B) Western blot showing the time course of p65 activation in HPSC and Panc1 cells. Cells were treated with recombinant DKK3 (10 μg/ml) for 0 to 24 hours, and changes in band density relative to baseline were quantified. (C) DKK3 stimulates NF-κB luciferase reporter in HPSC and PDAC cells, compared to the mutant luciferase reporter (MT). NF-κB activity induced by DKK3 was measured in Panc28 with phosphorylation-defective IκBαM by (D) luciferase reporter and (E) Western blotting. (F) Proliferation of Panc28 and Panc28/IκBαM was measured by MTT assay. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus PBS control.

Article Snippet: Primary rabbit anti-mouse DKK3 Ab was obtained from Proteintech Inc., goat anti-human DKK3 was obtained from Abcam, and rabbit anti-goat secondary Ab was obtained from Jackson ImmunoResearch Laboratories. mAbs against mouse α-SMA, Ki67, CD3, and CD8 were purchased from Abcam, Thermo Fisher Scientific, Santa Cruz Biotechnology, and BioLegend, respectively.

Techniques: Phospho-proteomics, Western Blot, Activation Assay, Recombinant, Luciferase, Mutagenesis, Activity Assay, MTT Assay, Control

Journal: Science translational medicine

Article Title: Suppression of stromal-derived Dickkopf-3 (DKK3) inhibits tumor progression and prolongs survival in pancreatic ductal adenocarcinoma

doi: 10.1126/scitranslmed.aat3487

Figure Lengend Snippet: BxPC3 tumor cells labeled with firefly luciferase were orthotopically implanted into nude mice, with or without either control HPSCs or HPSCs stably silenced for DKK3, in a 1:3 tumor/stroma ratio. (A) Average pancreas tumor volume and percentage of animals with metastases are shown at 35 days after injection. (B) IVIS imaging showing the growth of Panc02 tumor cells implanted subcutaneously in syngeneic C57/BL6 or DKK3-null mice, with Ki67 expression assessed by IHC. (C and D) Dkk3-deficient mice were crossed with KPC mice to produce P48-Cre; KrasLSL-G12D;Trp53fl/fl; Dkk3−/− progeny. Kaplan-Meier survival curve (C) and survival table (D) for mice with WT DKK3 (black), DKK3-null (red), or heterozygous DKK3 (blue). (E) Representative images of tumors from (C) from DKK3-WT mice (moribund, day 47), DKK3-heterozygous mice (moribund, day 63), or DKK3-null mice (early time point at day 48, or when moribund at day 68). (F) DKK3, (G) Ki67 proliferation index by IHC, and (H) collagen type I expression by qPCR. Data are means ± SEM [n = 8 to 15 mice per group in (A), 5 mice per group in (B), 10 to 16 mice per group in (C) to (D) and (F) to (H)]. *P < 0.05. Scale bars, 200 μm.

Article Snippet: Primary rabbit anti-mouse DKK3 Ab was obtained from Proteintech Inc., goat anti-human DKK3 was obtained from Abcam, and rabbit anti-goat secondary Ab was obtained from Jackson ImmunoResearch Laboratories. mAbs against mouse α-SMA, Ki67, CD3, and CD8 were purchased from Abcam, Thermo Fisher Scientific, Santa Cruz Biotechnology, and BioLegend, respectively.

Techniques: Labeling, Luciferase, Control, Stable Transfection, Injection, Imaging, Expressing

Journal: Science translational medicine

Article Title: Suppression of stromal-derived Dickkopf-3 (DKK3) inhibits tumor progression and prolongs survival in pancreatic ductal adenocarcinoma

doi: 10.1126/scitranslmed.aat3487

Figure Lengend Snippet: HPSCs and BxPC3 cells were treated with DKK3 mAb clones JM6-6-1 and JM8-12-1 or isotype control mAb or PBS. (A and B) HPSC apoptosis (A) and migration (B) as measured by fluorescence-activated cell sorting (FACS) and Transwell migration assay at 48 hours. (C) BxPC3 migration in response to rhDKK3 (10 μg/ml) as measured by Transwell migration assay at 48 hours. (D) BxPC3 resistance to gemcitabine (100 μM) as measured by MTT proliferation assay at 6 days. HPSC-CM (10 μg/ml). (E) The orthotopic coinjection BxPC3 + HPSC model of PDAC was used to test the efficacy of DKK3 mAb clones JM6-6-1 or JM8-12-1 (5 mg/kg ip, once every 5 days). Overall tumor progression was measured every 3 to 4 days by IVIS imaging. (F) Metastatic tumors in the peritoneal cavity after removal of the primary pancreatic tumor are shown by IVIS imaging. (G) Kaplan-Meier survival curve showing mice treated with DKK3 mAb clone JM8–12 (red), control Ab (blue), or PBS (black). KPC mice (P48-Cre; Kras LSL-G12D;Trp53fl/fl) either with WT DKK3 (solid lines) or deficient in DKK3 (dashed lines) were treated with DKK3 mAb JM6-6-1 (5 mg/kg, ip, once every 5 days), PBS, or control mAb. Kaplan-Meier survival curve (H) is shown with hazard ratios (log-rank test). Data are means ± SEM [n = 7 mice per group in (E) and (F), 6 to 7 mice per group in (G), 3 to 5 mice per group in (H)]. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus control Ab.

Article Snippet: Primary rabbit anti-mouse DKK3 Ab was obtained from Proteintech Inc., goat anti-human DKK3 was obtained from Abcam, and rabbit anti-goat secondary Ab was obtained from Jackson ImmunoResearch Laboratories. mAbs against mouse α-SMA, Ki67, CD3, and CD8 were purchased from Abcam, Thermo Fisher Scientific, Santa Cruz Biotechnology, and BioLegend, respectively.

Techniques: Clone Assay, Control, Migration, Fluorescence, FACS, Transwell Migration Assay, Proliferation Assay, Imaging

Journal: Science translational medicine

Article Title: Suppression of stromal-derived Dickkopf-3 (DKK3) inhibits tumor progression and prolongs survival in pancreatic ductal adenocarcinoma

doi: 10.1126/scitranslmed.aat3487

Figure Lengend Snippet: (A) T cells were stimulated and treated with DKK3 (5 to 10 μg/ml), and proliferation was measured by carboxyfluorescein diacetate succinimidyl ester (CFSE) assay. In a syngeneic orthotopic model with luciferase-labeled KPC cells, (B) tumors were examined for CD3 and CD8 expression by IHC, and (C) additional markers of T cell activity were measured by qPCR. (D) Mice in the syngeneic orthotopic KPC-luciferase model were treated with control IgG, DKK3 mAb JM6-6-1, α-CTLA4, or the combination JM6-6-1 + α-CTLA4, and tumor growth was measured by IVIS imaging up to 190 days. Survival in this orthotopic implantation model is shown in (E). Using a GEMM (F), KPC/DKK3+/+ (black) or KPC/DKK3−/− (blue) mice were treated with α-CTLA4 or control IgG, and the Kaplan-Meier survival curve is shown. Data are means ± SEM [n = 5 mice per group in (B) and (C), 5 to 7 mice per group in (D) and (E), 4 to 7 mice per group in (F)]. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars, 200 μm.

Article Snippet: Primary rabbit anti-mouse DKK3 Ab was obtained from Proteintech Inc., goat anti-human DKK3 was obtained from Abcam, and rabbit anti-goat secondary Ab was obtained from Jackson ImmunoResearch Laboratories. mAbs against mouse α-SMA, Ki67, CD3, and CD8 were purchased from Abcam, Thermo Fisher Scientific, Santa Cruz Biotechnology, and BioLegend, respectively.

Techniques: CFSE Assay, Luciferase, Labeling, Expressing, Activity Assay, Control, Imaging

Journal: Science translational medicine

Article Title: Suppression of stromal-derived Dickkopf-3 (DKK3) inhibits tumor progression and prolongs survival in pancreatic ductal adenocarcinoma

doi: 10.1126/scitranslmed.aat3487

Figure Lengend Snippet: Dkk3 expression was measured in HPSCs and PDAC cell lines by RTPCR (A) and qPCR (B) in monoculture and coculture. Striped bars indicate expression in HPSCs after coculture with PDAC cells. (C) Dkk3 expression in human PDAC and normal pancreatic tissue was determined by Affymetrix array. (D) IHC of DKK3 in a tissue microarray of human PDAC. Shown are representative fields from PDAC expressing low and high amounts of DKK3 with normal pancreas and negative controls. (E) DKK3 concentrations were measured by enzyme-linked immunosorbent assay (ELISA) in plasma samples from patients with PDAC, CP, or no pancreatic disease and in conditioned medium (CM) from HPSCs (HPSC-CM). (F) In a GEMM of PDAC, DKK3 is expressed early in development with CP and PanIN lesions and increases in PDAC. Scale bars, 200 μm. (G) Relative expression of Dkk3 in the GEMM of PDAC and in cancer cells isolated from GEMM tumors was quantified by Affymetrix. *P < 0.05, ***P < 0.001. Data are means ± SEM.

Article Snippet: Immunohistochemical analysis Primary rabbit anti-mouse DKK3 Ab was obtained from Proteintech Inc., goat anti-human DKK3 was obtained from Abcam, and rabbit anti-goat secondary Ab was obtained from Jackson ImmunoResearch Laboratories. mAbs against mouse α-SMA, Ki67, CD3, and CD8 were purchased from Abcam, Thermo Fisher Scientific, Santa Cruz Biotechnology, and BioLegend, respectively.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Microarray, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Isolation

Journal: Science translational medicine

Article Title: Suppression of stromal-derived Dickkopf-3 (DKK3) inhibits tumor progression and prolongs survival in pancreatic ductal adenocarcinoma

doi: 10.1126/scitranslmed.aat3487

Figure Lengend Snippet: (A) HPSC proliferation was measured by MTT [3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide] after treatment with PBS or rhDKK3 (10 μg/ml). OD490, optical density at 490 nm. DKK3 was silenced in HPSCs by shDKK3 (fig. S1B), cell proliferation was measured by MTT assay (B), and migration was determined at 24 hours (C). Control cells were transfected with scrambled short hairpin RNA (shRNA). (D) Panc1 cells were treated with rhDKK3 (10 μg/ml) or serum-free medium control, and cell migration and invasion were measured after 24 hours. (E) Panc1 cells were stably silenced for DKK3, and (E) cell proliferation and (F) colony formation in soft agar were measured. (G) BxPC3 cell migration was measured after treatment with CM from HPSCs or HPSCs silenced for DKK3. HPF, high-power field. (H) Soft agar colony formation in gemcitabine and (I) apoptosis were determined in chemosensitive L3.6pl cells expressing DKK3 compared with transfection controls. (J) Gemcitabine-induced apoptosis was measured in chemoresistant HS766T cells silenced for DKK3. *P < 0.01, **P < 0.001, ***P < 0.0001, ****P < 0.00001. Scale bars, 200 μm.

Article Snippet: Immunohistochemical analysis Primary rabbit anti-mouse DKK3 Ab was obtained from Proteintech Inc., goat anti-human DKK3 was obtained from Abcam, and rabbit anti-goat secondary Ab was obtained from Jackson ImmunoResearch Laboratories. mAbs against mouse α-SMA, Ki67, CD3, and CD8 were purchased from Abcam, Thermo Fisher Scientific, Santa Cruz Biotechnology, and BioLegend, respectively.

Techniques: MTT Assay, Migration, Control, Transfection, shRNA, Stable Transfection, Expressing

Journal: Science translational medicine

Article Title: Suppression of stromal-derived Dickkopf-3 (DKK3) inhibits tumor progression and prolongs survival in pancreatic ductal adenocarcinoma

doi: 10.1126/scitranslmed.aat3487

Figure Lengend Snippet: (A) Phosphorylation of p65 and IκBα induced by DKK3 treatment (10 μg/ml) was determined by Western blotting. Relative protein loading was shown by using anti–β-actin Ab. (B) Western blot showing the time course of p65 activation in HPSC and Panc1 cells. Cells were treated with recombinant DKK3 (10 μg/ml) for 0 to 24 hours, and changes in band density relative to baseline were quantified. (C) DKK3 stimulates NF-κB luciferase reporter in HPSC and PDAC cells, compared to the mutant luciferase reporter (MT). NF-κB activity induced by DKK3 was measured in Panc28 with phosphorylation-defective IκBαM by (D) luciferase reporter and (E) Western blotting. (F) Proliferation of Panc28 and Panc28/IκBαM was measured by MTT assay. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus PBS control.

Article Snippet: Immunohistochemical analysis Primary rabbit anti-mouse DKK3 Ab was obtained from Proteintech Inc., goat anti-human DKK3 was obtained from Abcam, and rabbit anti-goat secondary Ab was obtained from Jackson ImmunoResearch Laboratories. mAbs against mouse α-SMA, Ki67, CD3, and CD8 were purchased from Abcam, Thermo Fisher Scientific, Santa Cruz Biotechnology, and BioLegend, respectively.

Techniques: Phospho-proteomics, Western Blot, Activation Assay, Recombinant, Luciferase, Mutagenesis, Activity Assay, MTT Assay, Control

Journal: Science translational medicine

Article Title: Suppression of stromal-derived Dickkopf-3 (DKK3) inhibits tumor progression and prolongs survival in pancreatic ductal adenocarcinoma

doi: 10.1126/scitranslmed.aat3487

Figure Lengend Snippet: BxPC3 tumor cells labeled with firefly luciferase were orthotopically implanted into nude mice, with or without either control HPSCs or HPSCs stably silenced for DKK3, in a 1:3 tumor/stroma ratio. (A) Average pancreas tumor volume and percentage of animals with metastases are shown at 35 days after injection. (B) IVIS imaging showing the growth of Panc02 tumor cells implanted subcutaneously in syngeneic C57/BL6 or DKK3-null mice, with Ki67 expression assessed by IHC. (C and D) Dkk3-deficient mice were crossed with KPC mice to produce P48-Cre; KrasLSL-G12D;Trp53fl/fl; Dkk3−/− progeny. Kaplan-Meier survival curve (C) and survival table (D) for mice with WT DKK3 (black), DKK3-null (red), or heterozygous DKK3 (blue). (E) Representative images of tumors from (C) from DKK3-WT mice (moribund, day 47), DKK3-heterozygous mice (moribund, day 63), or DKK3-null mice (early time point at day 48, or when moribund at day 68). (F) DKK3, (G) Ki67 proliferation index by IHC, and (H) collagen type I expression by qPCR. Data are means ± SEM [n = 8 to 15 mice per group in (A), 5 mice per group in (B), 10 to 16 mice per group in (C) to (D) and (F) to (H)]. *P < 0.05. Scale bars, 200 μm.

Article Snippet: Immunohistochemical analysis Primary rabbit anti-mouse DKK3 Ab was obtained from Proteintech Inc., goat anti-human DKK3 was obtained from Abcam, and rabbit anti-goat secondary Ab was obtained from Jackson ImmunoResearch Laboratories. mAbs against mouse α-SMA, Ki67, CD3, and CD8 were purchased from Abcam, Thermo Fisher Scientific, Santa Cruz Biotechnology, and BioLegend, respectively.

Techniques: Labeling, Luciferase, Control, Stable Transfection, Injection, Imaging, Expressing

Journal: Science translational medicine

Article Title: Suppression of stromal-derived Dickkopf-3 (DKK3) inhibits tumor progression and prolongs survival in pancreatic ductal adenocarcinoma

doi: 10.1126/scitranslmed.aat3487

Figure Lengend Snippet: HPSCs and BxPC3 cells were treated with DKK3 mAb clones JM6-6-1 and JM8-12-1 or isotype control mAb or PBS. (A and B) HPSC apoptosis (A) and migration (B) as measured by fluorescence-activated cell sorting (FACS) and Transwell migration assay at 48 hours. (C) BxPC3 migration in response to rhDKK3 (10 μg/ml) as measured by Transwell migration assay at 48 hours. (D) BxPC3 resistance to gemcitabine (100 μM) as measured by MTT proliferation assay at 6 days. HPSC-CM (10 μg/ml). (E) The orthotopic coinjection BxPC3 + HPSC model of PDAC was used to test the efficacy of DKK3 mAb clones JM6-6-1 or JM8-12-1 (5 mg/kg ip, once every 5 days). Overall tumor progression was measured every 3 to 4 days by IVIS imaging. (F) Metastatic tumors in the peritoneal cavity after removal of the primary pancreatic tumor are shown by IVIS imaging. (G) Kaplan-Meier survival curve showing mice treated with DKK3 mAb clone JM8–12 (red), control Ab (blue), or PBS (black). KPC mice (P48-Cre; Kras LSL-G12D;Trp53fl/fl) either with WT DKK3 (solid lines) or deficient in DKK3 (dashed lines) were treated with DKK3 mAb JM6-6-1 (5 mg/kg, ip, once every 5 days), PBS, or control mAb. Kaplan-Meier survival curve (H) is shown with hazard ratios (log-rank test). Data are means ± SEM [n = 7 mice per group in (E) and (F), 6 to 7 mice per group in (G), 3 to 5 mice per group in (H)]. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus control Ab.

Article Snippet: Immunohistochemical analysis Primary rabbit anti-mouse DKK3 Ab was obtained from Proteintech Inc., goat anti-human DKK3 was obtained from Abcam, and rabbit anti-goat secondary Ab was obtained from Jackson ImmunoResearch Laboratories. mAbs against mouse α-SMA, Ki67, CD3, and CD8 were purchased from Abcam, Thermo Fisher Scientific, Santa Cruz Biotechnology, and BioLegend, respectively.

Techniques: Clone Assay, Control, Migration, Fluorescence, FACS, Transwell Migration Assay, Proliferation Assay, Imaging

Journal: Science translational medicine

Article Title: Suppression of stromal-derived Dickkopf-3 (DKK3) inhibits tumor progression and prolongs survival in pancreatic ductal adenocarcinoma

doi: 10.1126/scitranslmed.aat3487

Figure Lengend Snippet: (A) T cells were stimulated and treated with DKK3 (5 to 10 μg/ml), and proliferation was measured by carboxyfluorescein diacetate succinimidyl ester (CFSE) assay. In a syngeneic orthotopic model with luciferase-labeled KPC cells, (B) tumors were examined for CD3 and CD8 expression by IHC, and (C) additional markers of T cell activity were measured by qPCR. (D) Mice in the syngeneic orthotopic KPC-luciferase model were treated with control IgG, DKK3 mAb JM6-6-1, α-CTLA4, or the combination JM6-6-1 + α-CTLA4, and tumor growth was measured by IVIS imaging up to 190 days. Survival in this orthotopic implantation model is shown in (E). Using a GEMM (F), KPC/DKK3+/+ (black) or KPC/DKK3−/− (blue) mice were treated with α-CTLA4 or control IgG, and the Kaplan-Meier survival curve is shown. Data are means ± SEM [n = 5 mice per group in (B) and (C), 5 to 7 mice per group in (D) and (E), 4 to 7 mice per group in (F)]. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars, 200 μm.

Article Snippet: Immunohistochemical analysis Primary rabbit anti-mouse DKK3 Ab was obtained from Proteintech Inc., goat anti-human DKK3 was obtained from Abcam, and rabbit anti-goat secondary Ab was obtained from Jackson ImmunoResearch Laboratories. mAbs against mouse α-SMA, Ki67, CD3, and CD8 were purchased from Abcam, Thermo Fisher Scientific, Santa Cruz Biotechnology, and BioLegend, respectively.

Techniques: CFSE Assay, Luciferase, Labeling, Expressing, Activity Assay, Control, Imaging

Journal: Science translational medicine

Article Title: Suppression of stromal-derived Dickkopf-3 (DKK3) inhibits tumor progression and prolongs survival in pancreatic ductal adenocarcinoma

doi: 10.1126/scitranslmed.aat3487

Figure Lengend Snippet: Dkk3 expression was measured in HPSCs and PDAC cell lines by RTPCR (A) and qPCR (B) in monoculture and coculture. Striped bars indicate expression in HPSCs after coculture with PDAC cells. (C) Dkk3 expression in human PDAC and normal pancreatic tissue was determined by Affymetrix array. (D) IHC of DKK3 in a tissue microarray of human PDAC. Shown are representative fields from PDAC expressing low and high amounts of DKK3 with normal pancreas and negative controls. (E) DKK3 concentrations were measured by enzyme-linked immunosorbent assay (ELISA) in plasma samples from patients with PDAC, CP, or no pancreatic disease and in conditioned medium (CM) from HPSCs (HPSC-CM). (F) In a GEMM of PDAC, DKK3 is expressed early in development with CP and PanIN lesions and increases in PDAC. Scale bars, 200 μm. (G) Relative expression of Dkk3 in the GEMM of PDAC and in cancer cells isolated from GEMM tumors was quantified by Affymetrix. *P < 0.05, ***P < 0.001. Data are means ± SEM.

Article Snippet: Immunohistochemical analysis Primary rabbit anti-mouse DKK3 Ab was obtained from Proteintech Inc., goat anti-human DKK3 was obtained from Abcam, and rabbit anti-goat secondary Ab was obtained from Jackson ImmunoResearch Laboratories. mAbs against mouse α-SMA, Ki67, CD3, and CD8 were purchased from Abcam, Thermo Fisher Scientific, Santa Cruz Biotechnology, and BioLegend, respectively.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Microarray, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Isolation

Journal: Science translational medicine

Article Title: Suppression of stromal-derived Dickkopf-3 (DKK3) inhibits tumor progression and prolongs survival in pancreatic ductal adenocarcinoma

doi: 10.1126/scitranslmed.aat3487

Figure Lengend Snippet: (A) HPSC proliferation was measured by MTT [3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide] after treatment with PBS or rhDKK3 (10 μg/ml). OD490, optical density at 490 nm. DKK3 was silenced in HPSCs by shDKK3 (fig. S1B), cell proliferation was measured by MTT assay (B), and migration was determined at 24 hours (C). Control cells were transfected with scrambled short hairpin RNA (shRNA). (D) Panc1 cells were treated with rhDKK3 (10 μg/ml) or serum-free medium control, and cell migration and invasion were measured after 24 hours. (E) Panc1 cells were stably silenced for DKK3, and (E) cell proliferation and (F) colony formation in soft agar were measured. (G) BxPC3 cell migration was measured after treatment with CM from HPSCs or HPSCs silenced for DKK3. HPF, high-power field. (H) Soft agar colony formation in gemcitabine and (I) apoptosis were determined in chemosensitive L3.6pl cells expressing DKK3 compared with transfection controls. (J) Gemcitabine-induced apoptosis was measured in chemoresistant HS766T cells silenced for DKK3. *P < 0.01, **P < 0.001, ***P < 0.0001, ****P < 0.00001. Scale bars, 200 μm.

Article Snippet: Immunohistochemical analysis Primary rabbit anti-mouse DKK3 Ab was obtained from Proteintech Inc., goat anti-human DKK3 was obtained from Abcam, and rabbit anti-goat secondary Ab was obtained from Jackson ImmunoResearch Laboratories. mAbs against mouse α-SMA, Ki67, CD3, and CD8 were purchased from Abcam, Thermo Fisher Scientific, Santa Cruz Biotechnology, and BioLegend, respectively.

Techniques: MTT Assay, Migration, Control, Transfection, shRNA, Stable Transfection, Expressing

Journal: Science translational medicine

Article Title: Suppression of stromal-derived Dickkopf-3 (DKK3) inhibits tumor progression and prolongs survival in pancreatic ductal adenocarcinoma

doi: 10.1126/scitranslmed.aat3487

Figure Lengend Snippet: (A) Phosphorylation of p65 and IκBα induced by DKK3 treatment (10 μg/ml) was determined by Western blotting. Relative protein loading was shown by using anti–β-actin Ab. (B) Western blot showing the time course of p65 activation in HPSC and Panc1 cells. Cells were treated with recombinant DKK3 (10 μg/ml) for 0 to 24 hours, and changes in band density relative to baseline were quantified. (C) DKK3 stimulates NF-κB luciferase reporter in HPSC and PDAC cells, compared to the mutant luciferase reporter (MT). NF-κB activity induced by DKK3 was measured in Panc28 with phosphorylation-defective IκBαM by (D) luciferase reporter and (E) Western blotting. (F) Proliferation of Panc28 and Panc28/IκBαM was measured by MTT assay. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus PBS control.

Article Snippet: Immunohistochemical analysis Primary rabbit anti-mouse DKK3 Ab was obtained from Proteintech Inc., goat anti-human DKK3 was obtained from Abcam, and rabbit anti-goat secondary Ab was obtained from Jackson ImmunoResearch Laboratories. mAbs against mouse α-SMA, Ki67, CD3, and CD8 were purchased from Abcam, Thermo Fisher Scientific, Santa Cruz Biotechnology, and BioLegend, respectively.

Techniques: Phospho-proteomics, Western Blot, Activation Assay, Recombinant, Luciferase, Mutagenesis, Activity Assay, MTT Assay, Control

Journal: Science translational medicine

Article Title: Suppression of stromal-derived Dickkopf-3 (DKK3) inhibits tumor progression and prolongs survival in pancreatic ductal adenocarcinoma

doi: 10.1126/scitranslmed.aat3487

Figure Lengend Snippet: BxPC3 tumor cells labeled with firefly luciferase were orthotopically implanted into nude mice, with or without either control HPSCs or HPSCs stably silenced for DKK3, in a 1:3 tumor/stroma ratio. (A) Average pancreas tumor volume and percentage of animals with metastases are shown at 35 days after injection. (B) IVIS imaging showing the growth of Panc02 tumor cells implanted subcutaneously in syngeneic C57/BL6 or DKK3-null mice, with Ki67 expression assessed by IHC. (C and D) Dkk3-deficient mice were crossed with KPC mice to produce P48-Cre; KrasLSL-G12D;Trp53fl/fl; Dkk3−/− progeny. Kaplan-Meier survival curve (C) and survival table (D) for mice with WT DKK3 (black), DKK3-null (red), or heterozygous DKK3 (blue). (E) Representative images of tumors from (C) from DKK3-WT mice (moribund, day 47), DKK3-heterozygous mice (moribund, day 63), or DKK3-null mice (early time point at day 48, or when moribund at day 68). (F) DKK3, (G) Ki67 proliferation index by IHC, and (H) collagen type I expression by qPCR. Data are means ± SEM [n = 8 to 15 mice per group in (A), 5 mice per group in (B), 10 to 16 mice per group in (C) to (D) and (F) to (H)]. *P < 0.05. Scale bars, 200 μm.

Article Snippet: Immunohistochemical analysis Primary rabbit anti-mouse DKK3 Ab was obtained from Proteintech Inc., goat anti-human DKK3 was obtained from Abcam, and rabbit anti-goat secondary Ab was obtained from Jackson ImmunoResearch Laboratories. mAbs against mouse α-SMA, Ki67, CD3, and CD8 were purchased from Abcam, Thermo Fisher Scientific, Santa Cruz Biotechnology, and BioLegend, respectively.

Techniques: Labeling, Luciferase, Control, Stable Transfection, Injection, Imaging, Expressing

Journal: Science translational medicine

Article Title: Suppression of stromal-derived Dickkopf-3 (DKK3) inhibits tumor progression and prolongs survival in pancreatic ductal adenocarcinoma

doi: 10.1126/scitranslmed.aat3487

Figure Lengend Snippet: HPSCs and BxPC3 cells were treated with DKK3 mAb clones JM6-6-1 and JM8-12-1 or isotype control mAb or PBS. (A and B) HPSC apoptosis (A) and migration (B) as measured by fluorescence-activated cell sorting (FACS) and Transwell migration assay at 48 hours. (C) BxPC3 migration in response to rhDKK3 (10 μg/ml) as measured by Transwell migration assay at 48 hours. (D) BxPC3 resistance to gemcitabine (100 μM) as measured by MTT proliferation assay at 6 days. HPSC-CM (10 μg/ml). (E) The orthotopic coinjection BxPC3 + HPSC model of PDAC was used to test the efficacy of DKK3 mAb clones JM6-6-1 or JM8-12-1 (5 mg/kg ip, once every 5 days). Overall tumor progression was measured every 3 to 4 days by IVIS imaging. (F) Metastatic tumors in the peritoneal cavity after removal of the primary pancreatic tumor are shown by IVIS imaging. (G) Kaplan-Meier survival curve showing mice treated with DKK3 mAb clone JM8–12 (red), control Ab (blue), or PBS (black). KPC mice (P48-Cre; Kras LSL-G12D;Trp53fl/fl) either with WT DKK3 (solid lines) or deficient in DKK3 (dashed lines) were treated with DKK3 mAb JM6-6-1 (5 mg/kg, ip, once every 5 days), PBS, or control mAb. Kaplan-Meier survival curve (H) is shown with hazard ratios (log-rank test). Data are means ± SEM [n = 7 mice per group in (E) and (F), 6 to 7 mice per group in (G), 3 to 5 mice per group in (H)]. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus control Ab.

Article Snippet: Immunohistochemical analysis Primary rabbit anti-mouse DKK3 Ab was obtained from Proteintech Inc., goat anti-human DKK3 was obtained from Abcam, and rabbit anti-goat secondary Ab was obtained from Jackson ImmunoResearch Laboratories. mAbs against mouse α-SMA, Ki67, CD3, and CD8 were purchased from Abcam, Thermo Fisher Scientific, Santa Cruz Biotechnology, and BioLegend, respectively.

Techniques: Clone Assay, Control, Migration, Fluorescence, FACS, Transwell Migration Assay, Proliferation Assay, Imaging

Journal: Science translational medicine

Article Title: Suppression of stromal-derived Dickkopf-3 (DKK3) inhibits tumor progression and prolongs survival in pancreatic ductal adenocarcinoma

doi: 10.1126/scitranslmed.aat3487

Figure Lengend Snippet: (A) T cells were stimulated and treated with DKK3 (5 to 10 μg/ml), and proliferation was measured by carboxyfluorescein diacetate succinimidyl ester (CFSE) assay. In a syngeneic orthotopic model with luciferase-labeled KPC cells, (B) tumors were examined for CD3 and CD8 expression by IHC, and (C) additional markers of T cell activity were measured by qPCR. (D) Mice in the syngeneic orthotopic KPC-luciferase model were treated with control IgG, DKK3 mAb JM6-6-1, α-CTLA4, or the combination JM6-6-1 + α-CTLA4, and tumor growth was measured by IVIS imaging up to 190 days. Survival in this orthotopic implantation model is shown in (E). Using a GEMM (F), KPC/DKK3+/+ (black) or KPC/DKK3−/− (blue) mice were treated with α-CTLA4 or control IgG, and the Kaplan-Meier survival curve is shown. Data are means ± SEM [n = 5 mice per group in (B) and (C), 5 to 7 mice per group in (D) and (E), 4 to 7 mice per group in (F)]. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars, 200 μm.

Article Snippet: Immunohistochemical analysis Primary rabbit anti-mouse DKK3 Ab was obtained from Proteintech Inc., goat anti-human DKK3 was obtained from Abcam, and rabbit anti-goat secondary Ab was obtained from Jackson ImmunoResearch Laboratories. mAbs against mouse α-SMA, Ki67, CD3, and CD8 were purchased from Abcam, Thermo Fisher Scientific, Santa Cruz Biotechnology, and BioLegend, respectively.

Techniques: CFSE Assay, Luciferase, Labeling, Expressing, Activity Assay, Control, Imaging