antibody against fzd7  (R&D Systems)

Journal: Nature Communications

Article Title: Deep learning-assisted discovery of a potent and cell-active inhibitor of RNA N 6 -methyladenosine recognition protein YTHDC2

doi: 10.1038/s41467-025-65542-0

Figure Lengend Snippet: a Western blot analyses of H3K4me3 expression in DC2-C1 -treated PANC-1 or MiaPACA-2 cells. Blots shown are representative of three biological replicates. Full blots can be found in Supplementary Fig. . b Quantitative analysis of Western blot for PANC-1 or MiaPACA-2 cells. Data are shown as the mean ± SD from three technical replicates in three independent experiments. c Colony formation assay of PANC-1 or MiaPACA-2 cells treated with DC2-C1 or 16o . d Abilities of invasion of PANC-1 or MiaPACA-2 cells treated with DC2-C1 or 16o . Scale bar, 50 μm. e-g ACC1 , FASN and SREBP-1C mRNA expression were determined by RT-qPCR in DC2-C1 -treated HepG2 cells. Data are shown as the mean ± SD from three technical replicates in three independent experiments. h Western blot analyses of ID2 expression in DC2-C1 -treated PANC-1 cells; Western blot analyses of FZD7 expression in DC2-C1 -treated HGC27 cells. Blots shown are representative of three biological replicates. Full blots can be found in Supplementary Fig. . i FZD7 mRNA expression was determined by RT-qPCR in DC2-C1 -treated HGC27 cells. Data are shown as the mean ± SD from four technical replicates in four independent experiments. j ID2 mRNA expression was validated by RT-qPCR in DC2-C1 -treated PANC-1 cells. Data are shown as the mean ± SD from four technical replicates in four independent experiments. Source data are provided as a Source Data file. (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; by two-tailed unpaired Student's t-test).

Article Snippet: Antibodies used: YTHDC2 antibody (ZenBio, R27443 ), AMIGO2 antibody (ImmunoWay, YN2372), ID2 antibody (HUABIO, M1301-2), H3K4me3 antibody (Abcam, ab8580), FZD7 antibody (Proteintech, 16974-1-AP), DYKDDDDK Tag (Flag-tag) antibody (Cell Signaling Technology, 14793), GAPDH antibody (Proteintech, 60004-1-Ig), HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H + L) (Proteintech, SA00001-2), HRP-conjugated Affinipure Goat Anti-Mouse IgG(H + L) (Proteintech, SA00001-1).

Techniques: Western Blot, Expressing, Colony Assay, Quantitative RT-PCR, Two Tailed Test

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: A novel Frizzled 7 antibody disrupts the Wnt pathway and inhibits Wilms tumor growth

doi: 10.3389/fbioe.2025.1641137

Figure Lengend Snippet: Schematic representation of the FZD7 protein structure and its localization within the cell membrane. Four peptides were selected for antibody generation; clones 288.1, 288.2, and 288.5 derived from peptide 4 were subsequently chosen for detailed analysis. Figure created with BioRender.com.

Article Snippet: The cells were stained with an isotype control (R and D Systems Cat# IC006A, RRID:AB_357254) or a primary antibody against FZD7 (R and D Systems Cat# FAB1981A, RRID:AB_2232278).

Techniques: Membrane, Clone Assay, Derivative Assay

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: A novel Frizzled 7 antibody disrupts the Wnt pathway and inhibits Wilms tumor growth

doi: 10.3389/fbioe.2025.1641137

Figure Lengend Snippet: Binding of FZD7 antibody to Wilms tumor cells, expression analysis of different WT donors and effect on cell proliferation. (A) Percentage of Frizzle 7 (FZD7)-expressing cells in culture. A representative flow cytometry analysis is shown for primary Wilms tumor (WT)-derived cells (upper) and WT-xenograft derived cells (lower). (B) A summarizing bar graph of FZD7 expression in WT cells from different donors used for in vitro experiments. (C) Effect of secreted antibodies on WT cells: Relative percentage of viable cells following 48 h of treatment with the different clones. The spent medium served as a control. The numbers indicate the hybridoma clone from which the spent medium was derived; *p < 0.05, **p < 0.01. The results are shown as the mean ± SEM of at least three different experiments for each antibody.

Article Snippet: The cells were stained with an isotype control (R and D Systems Cat# IC006A, RRID:AB_357254) or a primary antibody against FZD7 (R and D Systems Cat# FAB1981A, RRID:AB_2232278).

Techniques: Binding Assay, Wilms Tumor Assay, Expressing, Flow Cytometry, Derivative Assay, In Vitro, Clone Assay, Control

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: A novel Frizzled 7 antibody disrupts the Wnt pathway and inhibits Wilms tumor growth

doi: 10.3389/fbioe.2025.1641137

Figure Lengend Snippet: Binding characterization of FZD7 -288.1 in Immune Precipitation assays, Western blot analysis, FACS and Immunohistochemistry. (A) αFZD7-288.1 but not 288.5 immunoprecipitated the Frizzled 7 (FZD7) protein. A protein immunoprecipitation (IP) assay was used to validate the antibody-specific interaction with the FZD7 receptor. SK-MEL28 total cell lysates (FZD7 Input) and FZD7-bound fractions (FZD7 IP) were assayed via Western blot analysis. For detection, a commercial anti-FZD7 antibody (Millipore) was used. Nonspecific antibody and mouse IgG served as negative controls, and β-actin was used as a loading control. A representative blot of three separate experiments is shown. (B) Specific binding to the FZD7 receptor was examined in HEK293T cells overexpressing full-length FZD7. The cell extracts were probed with different concentrations of αFZD7-288.1. Specific binding of the Ab to FZD7-expressing cells was observed. (C) Probing of cell lysates from sh-FZD7-Wilms tumor (WT) cells with αFZD7-288.1 revealed the downregulation of FZD7, similar to the effect of a commercial αFZD7 antibody, demonstrating its specificity. β-Actin was used as a loading control. (D) Representative flow cytometry analysis of FZD7 expression in cultured WT cells stained with both αFZD7-288.1 and a rat anti-FZD7 APC-conjugated antibody. Cells stained positive for αFZD7-288.1 were mostly observed as a subpopulation within the cells detected by RαFZD7-APC. The data are presented in a dot plot graph showing FZD7 staining in red and isotype control staining (negative control) in gray.

Article Snippet: The cells were stained with an isotype control (R and D Systems Cat# IC006A, RRID:AB_357254) or a primary antibody against FZD7 (R and D Systems Cat# FAB1981A, RRID:AB_2232278).

Techniques: Binding Assay, Western Blot, Immunohistochemistry, Immunoprecipitation, Control, Expressing, Wilms Tumor Assay, Flow Cytometry, Cell Culture, Staining, Negative Control

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: A novel Frizzled 7 antibody disrupts the Wnt pathway and inhibits Wilms tumor growth

doi: 10.3389/fbioe.2025.1641137

Figure Lengend Snippet: The αFZD7-288.1 antibody inhibits β-catenin translocating Wnt signaling in Wilms tumor cells. (A) Western blot (WB) analysis demonstrating the inhibition of active β-catenin and decreased levels of Fizzled 7 (FZD7). (B) following treatment with 5 μg/mL αFZD7-288.1 for 48 h in Wilms tumor (WT) cells. A nonspecific Ab was used as a negative control; β-actin was used as a loading control. (C) β-catenin immunofluorescence staining of untreated WT and αFZD7-288.1-treated cells: Double-staining with DAPI and cytokeratin (first panel) and with cytokeratin and β-catenin (second panel). β-catenin staining revealed strong expression and a greater percentage of cells with nuclear localization of β-catenin in the control untreated cells (scale bar represents 50 µM) X70 magnification of representative cells (indicated by arrows) from each group (third panel) and double-staining with DAPI and β-catenin (forth panel). (D–F) Real-time PCR gene expression analysis of WT cells: The expression of Wnt transducers [ (D) ; FZD7, β-catenin], Wnt pathway target genes [ (E) ; AXIN2, CCND1, MYC-C], and Wnt pathway inhibitors [ (F) ; DKK1, sFRP1] was also measured. The results showed reduced mRNA expression of Wnt pathway-related genes. The data are shown as the mean ± S.E.M. of 5 separate experiments; *p < 0.05; **p < 0.01.

Article Snippet: The cells were stained with an isotype control (R and D Systems Cat# IC006A, RRID:AB_357254) or a primary antibody against FZD7 (R and D Systems Cat# FAB1981A, RRID:AB_2232278).

Techniques: Wilms Tumor Assay, Western Blot, Inhibition, Negative Control, Control, Immunofluorescence, Staining, Double Staining, Expressing, Real-time Polymerase Chain Reaction, Gene Expression

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: A novel Frizzled 7 antibody disrupts the Wnt pathway and inhibits Wilms tumor growth

doi: 10.3389/fbioe.2025.1641137

Figure Lengend Snippet: The αFZD7-288.1 antibody reduces proliferation in Wilms tumors cells and induces cell death. (A) Inhibition of the Wnt pathway by αFZD7-288.1 treatment induced changes in cell kinetics, as demonstrated by the longer doubling time of antibody treated Wilms tumor (WT) cells compared to control treated WT cells and (B) the significantly lower growth rate (defined as the number of cell divisions per day), *p < 0.05; n = 4. Reduced proliferation in cells treated with αFZD7-288.1 was also observed by MTS assay in (C) and (D) Quantification of Ki67 staining was performed by counting the number of Ki67-positive nuclei in five high-power fields (HPF) per condition, using representative regions from each sample. Reduced nuclear Ki67 expression (red, lower panels) compared to that in control untreated cells (magnification ×40 scale bars = 100 μm; magnification ×20 scale bars = 50 µm). (E) Induction of cell death in WT cells is shown in a representative analysis for annexin V staining, which demonstrated a marked increase in the percentage of apoptotic and preapoptotic cells. (F) Percentage of dead cells calculated using trypan blue staining of WT cells indicating significantly greater cell death in treated WT cells than in control cells; *p < 0.05; n = 4.

Article Snippet: The cells were stained with an isotype control (R and D Systems Cat# IC006A, RRID:AB_357254) or a primary antibody against FZD7 (R and D Systems Cat# FAB1981A, RRID:AB_2232278).

Techniques: Inhibition, Wilms Tumor Assay, Control, MTS Assay, Staining, Expressing

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: A novel Frizzled 7 antibody disrupts the Wnt pathway and inhibits Wilms tumor growth

doi: 10.3389/fbioe.2025.1641137

Figure Lengend Snippet: The migration and sphere formation of Wilms tumor cells is reduced after treatment with αFZD7-288.1 antibody. (A) Scratch assay demonstrating the effects of αFZD7-288.1 on the ability of cells to migrate through a scrape in the cell monolayer. Wilms tumor (WT)-treated cells demonstrated a lower migration capacity than control cells, as shown in the representative images. Calculation of the area of migrating cells (on the right) showed that the cells had a significantly reduced migration capacity following treatment; *p < 0.05; n = 3. (B) A sphere formation assay was performed. The number and size of the spheres formed by the treated cells were significantly lower than those formed by the untreated cells. The data are presented as the means ± SEMs of at least three experiments performed in triplicate (*p < 0.05) On the left, representative phase-contrast images of spheres formed by treated and untreated WT cells showing markedly fewer condensed spheres in the treated cells (scale bars = 100 μm, magnification ×10).

Article Snippet: The cells were stained with an isotype control (R and D Systems Cat# IC006A, RRID:AB_357254) or a primary antibody against FZD7 (R and D Systems Cat# FAB1981A, RRID:AB_2232278).

Techniques: Migration, Wilms Tumor Assay, Wound Healing Assay, Control, Tube Formation Assay

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: A novel Frizzled 7 antibody disrupts the Wnt pathway and inhibits Wilms tumor growth

doi: 10.3389/fbioe.2025.1641137

Figure Lengend Snippet: αFZD7-288.1 strongly co-localizes with NCAM1 in WT, and treatment with αFZD7-288.1 reduces WT CSC markers. (A) Immunofluorescence of staining of cultured WT cells using αFZD7-288.1 and NCAM1 and co-staining (αFZD7-red, αNCAM1 green and DAPI blue) microscopy revealed overlapping signals as a merged color (yellow), indicating membrane spatial proximity. Slides were imaged using Olympus IX83 fluorescent microscope and Olympus DP80 camera. Image processing was done using Olympus OlyVia software. (B) The expression of the Cancer stem cell marker genes NCAM1, CITED, SIX2 and ALDH1A1 decreased in the antibody αFZD7-288.1 treated WT derived cells (n = 3); *p < 0.05; **p < 0.01.

Article Snippet: The cells were stained with an isotype control (R and D Systems Cat# IC006A, RRID:AB_357254) or a primary antibody against FZD7 (R and D Systems Cat# FAB1981A, RRID:AB_2232278).

Techniques: Immunofluorescence, Staining, Cell Culture, Microscopy, Membrane, Software, Expressing, Marker, Derivative Assay

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: A novel Frizzled 7 antibody disrupts the Wnt pathway and inhibits Wilms tumor growth

doi: 10.3389/fbioe.2025.1641137

Figure Lengend Snippet: The antitumor activity of αFZD7-288.1 as demonstrated in changes in tumor volumes. (A) Flow cytometry histogram evaluating Frizzled 7 (FZD7) expression in Wilms tumor (WT) tissue for use in vivo . (B) Workflow diagram illustrating the in vivo experimental design. (C) Mouse weights during treatment. Paclitaxel (PTX)-treated mice suffered from toxic side effects and significant weight loss following treatment; **p < 0.01; ***p < 0.005. (D) Antitumor activity: Mice were treated intravenously with normal saline, αFZD7-288.1 (10 mg/kg) or paclitaxel (15 mg/kg). Tumor volume, presented in mm 3 , was assessed every 2–3 days using an external electronic caliper and calculated by the modified ellipsoid formula V = 0.52 × (length × width 2 ). Like PTX-treated tumors, tumors treated with αFZD7-288.1 exhibited growth inhibition and even tumor shrinkage, while saline-treated tumors showed rapid tumor growth; *p < 0.05; **p < 0.01. (E) Representative images on day 10 of the experiment showing mice with large saline-treated tumors (right) and unnoticeable αFZD7-288.1-treated tumors (left). (F) Waterfall graph showing the changes in the individual tumor volumes with respect to the initial tumor size. (G) FZD7 expression in tumors treated with αFZD7-288.1 was significantly altered by 0.445-fold; *p < 0.05; n = 3 per group. (H) The expression of the canonical Wnt pathway genes FZD7, β-CATENIN, AXIN2, CCND1, C-MYC, DKK1, and sFRP1 decreased in the treated tumors (n = 4); *p < 0.05; **p < 0.01; ***p < 0.005.

Article Snippet: The cells were stained with an isotype control (R and D Systems Cat# IC006A, RRID:AB_357254) or a primary antibody against FZD7 (R and D Systems Cat# FAB1981A, RRID:AB_2232278).

Techniques: Activity Assay, Flow Cytometry, Expressing, Wilms Tumor Assay, In Vivo, Saline, Modification, Inhibition

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: A novel Frizzled 7 antibody disrupts the Wnt pathway and inhibits Wilms tumor growth

doi: 10.3389/fbioe.2025.1641137

Figure Lengend Snippet: The antitumor activity of αFZD7-288.1 as demonstrated by proliferative markers in Immunohistochemical staining. (A) Immunohistochemical staining for Ki67 and Caspase3 in representative tumors from the different experimental groups; saline, paclitaxel (PTX) and αFZD7-288.1 (type A, representing reduced-volume tumors; and type B, representing tumors with growth inhibition compared to saline-treated tumors). The scale bars represent 50 μm at a magnification of ×20. Compared with those of saline- or PTX-treated tumors, αFZD7-288.1-treated tumors exhibited reduced proliferation. An increase in apoptosis was observed in αFZD7-288.1- and PTX-treated tumors (B) .

Article Snippet: The cells were stained with an isotype control (R and D Systems Cat# IC006A, RRID:AB_357254) or a primary antibody against FZD7 (R and D Systems Cat# FAB1981A, RRID:AB_2232278).

Techniques: Activity Assay, Immunohistochemical staining, Staining, Saline, Inhibition