human fzd7  (Proteintech)

Journal: PLOS Biology

Article Title: Targeted inhibition of Wnt signaling with a Clostridioides difficile toxin B fragment suppresses breast cancer tumor growth

doi: 10.1371/journal.pbio.3002353

Figure Lengend Snippet: (A) Representative immunostaining images detecting the levels of active β-catenin (non-phosphorylated form), K14, K8 in tumor tissues from Trp53/Brca1 (p53/BRCA1)-deficient, MMTV-PyMT and C3(1)-Tag breast cancer mouse models. DAPI staining marks the cell nucleus. Scale bar = 50 μm. (B) Quantitation of active β-catenin positive cells in (A). (C) Immunoblot analysis of active β-catenin expression in mammary tumors from models described in (A and B). Actin serves as a loading control. (D) qRT-PCR analysis of expression of Fzd family genes in p53/BRCA1-deficient ( n = 3), C3(1)-Tag ( n = 3), and MMTV-PyMT ( n = 2) tumors; error bars indicate mean ± SEM. (E) Upper plots: FACS analysis using antibodies against CD24 and CD29 to differentiate basal versus luminal normal MECs in female mice; lower plots: FACS analysis using a FZD7-specific antibody identified FZD7 + in Lin- (negative for lineage markers, CD45, CD31, TER119) cells and in luminal and basal MEC populations. (F) The percentages of FZD7 + cells in p53/BRCA1-deficient, C3(1)-Tag and MMTV-PyMT tumors, as well as in normal mouse mammary glands, were analyzed by FACS. The FZD7 + peaks are marked and compared in the upper panel and quantified in the lower panel (the P values for p53/BRCA1-deficient, C3(1)-Tag and MMTV-PyMT models were <0.0001, 0.8556, 0.0178, respectively, compared to normal mouse mammary glands). The representative FACS plots are shown in . Numerical values are in . FACS, fluorescence-activated cell sorting; MEC, mammary epithelial cell; qRT-PCR, quantitative real-time PCR.

Article Snippet: The following antibodies were utilized: CD24 (clone M1/69, 564237; BD Biosciences; 1:100), CD29 (clone eBioHMb1-1, 12-0291-82; 1:250), FITC-TcdB FBD (0.1 mg/ml), FZD7 (Clone 151143, FAB1981A; RD system; 1:100), CD31 (clone 390, 13-0311-85; eBioscience 1:100), CD45 (clone 30-F11, 13-0451-82; eBioscience; 1:100), and TER119 (clone Ter-119, 13-5921-85; eBioscience; 1:100).

Techniques: Immunostaining, Staining, Quantitation Assay, Western Blot, Expressing, Control, Quantitative RT-PCR, Fluorescence, FACS, Real-time Polymerase Chain Reaction

Journal: PLOS Biology

Article Title: Targeted inhibition of Wnt signaling with a Clostridioides difficile toxin B fragment suppresses breast cancer tumor growth

doi: 10.1371/journal.pbio.3002353

Figure Lengend Snippet: (A, B) Tumor organoid cells from p53/BRAC1-deficient tumor model (A) or C3(1)-Tag model (B) were injected into nude mice, and mice were then treated with TcdB FBD alone, cisplatin alone, or a combination of both TcdB FBD and cisplatin as indicated. Tumor volumes were recorded and plotted over time. Error bars indicate mean ± SEM, n = 8 mice. (C) FACS analysis of FZD7 + cells in the indicated tumors from p53/BRCA1-deficient ( n = 5) and C3(1)-Tag ( n = 4) models; the FACS gating strategy is the same as those shown in . Error bars indicate mean ± SEM. (D) Tumor organoid cells from MMTV-PyMT model were injected into nude mice, and mice were then treated with TcdB FBD alone, cisplatin alone, or a combination of both TcdB FBD and cisplatin as indicated. Tumor volumes were recorded and plotted over time. Error bars indicate mean ± SEM, n = 6–8 mice. (E) Representative images of MMTV-PyMT mammary tumor organoids treated with TcdB FBD alone (150 nM), cisplatin alone (0.2 μM), or a combination of cisplatin and TcdB FBD . Scale bar = 100 μm. (F) Quantitation of organoid sizes for indicated groups described in (E). (G) Quantitation of organoid numbers for indicated groups described in (E). (H) qRT-PCR analysis of EMT-related genes ( Vim , Zeb1 , Tcf4 ) and Fzd7 genes in tumor cells from MMTV-PyMT model after treatment with TcdB FBD , cisplatin, or a combination of both ( P < 0.01). Numerical values are in . EMT, epithelial–mesenchymal transition; FACS, fluorescence-activated cell sorting; qRT-PCR, quantitative real-time PCR.

Article Snippet: The following antibodies were utilized: CD24 (clone M1/69, 564237; BD Biosciences; 1:100), CD29 (clone eBioHMb1-1, 12-0291-82; 1:250), FITC-TcdB FBD (0.1 mg/ml), FZD7 (Clone 151143, FAB1981A; RD system; 1:100), CD31 (clone 390, 13-0311-85; eBioscience 1:100), CD45 (clone 30-F11, 13-0451-82; eBioscience; 1:100), and TER119 (clone Ter-119, 13-5921-85; eBioscience; 1:100).

Techniques: Injection, Quantitation Assay, Quantitative RT-PCR, Fluorescence, FACS, Real-time Polymerase Chain Reaction

Journal: ACS Omega

Article Title: Conjugation of Antibodies and siRNA Duplexes to Polymer Nanoparticles via Maleimide–Thiol Chemistry

doi: 10.1021/acsomega.4c07025

Figure Lengend Snippet: Characterization of modified antibodies. (a) Quantification of free thiols present in antibodies treated with TCEP via Ellman’s assay ( n = 3). Data are the mean ± standard deviation. (b) SDS-PAGE of unmodified anti-FZD7 antibodies, anti-FZD7 antibodies treated with TCEP (i.e., FZD7-SH), and anti-FZD7 antibodies conjugated to an OPSS-PEG-SVA linker (i.e., FZD7-linker). (c) Quantification of antibody binding avidity to MDA-MB-231 cells using an ELISA. Relative binding was normalized to that of the unmodified anti-FZD7 antibodies ( n = 3). Data are mean ± standard deviation.

Article Snippet: Either normal rabbit IgG (2729S, Cell Signaling Technology) or rabbit anti-human FZD7 antibodies (LS-C383580, LSBio) were used.

Techniques: Modification, Standard Deviation, SDS Page, Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: ACS Omega

Article Title: Conjugation of Antibodies and siRNA Duplexes to Polymer Nanoparticles via Maleimide–Thiol Chemistry

doi: 10.1021/acsomega.4c07025

Figure Lengend Snippet: Synthesis and characterization of antibody-conjugated NPs. (a) Simplified protocol for conjugation of modified FZD7 antibodies to PLGA NPs. (b) Mode diameter and mean zeta potential of NPs conjugated with FZD7 antibodies ( n = 3). Error bars indicate the standard deviation.

Article Snippet: Either normal rabbit IgG (2729S, Cell Signaling Technology) or rabbit anti-human FZD7 antibodies (LS-C383580, LSBio) were used.

Techniques: Conjugation Assay, Modification, Zeta Potential Analyzer, Standard Deviation

Journal: ACS Omega

Article Title: Conjugation of Antibodies and siRNA Duplexes to Polymer Nanoparticles via Maleimide–Thiol Chemistry

doi: 10.1021/acsomega.4c07025

Figure Lengend Snippet: Synthesis and characterization of NPs modified with both antibodies and siRNAs. (a) Simplified protocol for coconjugation of FZD7-linker antibodies and siβcat siRNAs. (b) Mode diameter and zeta potential of NPs conjugated with both FZD7-linker antibodies and siβcat siRNAs ( n = 3). Error bars indicate standard deviation.

Article Snippet: Either normal rabbit IgG (2729S, Cell Signaling Technology) or rabbit anti-human FZD7 antibodies (LS-C383580, LSBio) were used.

Techniques: Modification, Zeta Potential Analyzer, Standard Deviation