Journal: Cell Death & Disease

Article Title: Long non-coding RNA DSCR8 acts as a molecular sponge for miR-485-5p to activate Wnt/β-catenin signal pathway in hepatocellular carcinoma

doi: 10.1038/s41419-018-0937-7

Figure Lengend Snippet: a Data from bioinformatics tools (microRNA.org, TargetScan, and miRDB) showed that there were putative binding sites between 3′UTR of FZD7-wt and miR-485-5p. FZD7-mut means mutation of binding sites in 3′UTR of FZD7. b Luciferase reporter gene assays revealed that miR-485-5p negatively regulated the luciferase activity of FZD7-wt-3′UTR, rather than of DSCR8-mut-3′UTR. The mRNA ( c , d ) and protein ( e , f ) expression of FZD7 was negatively regulated by miR-485-5p. And FZD7 clone or siRNAs reversed the effects of miR-485-5p mimics or inhibitors on FZD7 expression ( c – f ). e , f Western blot results revealed that the accumulation of cytoplasmic β-catenin and the accumulation of nuclear β-catenin, c-Myc expression, and cyclin D1 expression were negatively regulated by miR-485-5p, while reversed by FZD7 clone and siRNA. n = three repeats with similar results, ** P < 0.01, *** P < 0.001

Article Snippet: FZD7 Human cDNA ORF Clone (FZD7) and FZD7 siRNA (si-FZD7) were purchased from OriGene (OriGene Technologies,Inc., USA).

Techniques: Binding Assay, Mutagenesis, Luciferase, Activity Assay, Expressing, Western Blot

Journal: Cell Death & Disease

Article Title: Long non-coding RNA DSCR8 acts as a molecular sponge for miR-485-5p to activate Wnt/β-catenin signal pathway in hepatocellular carcinoma

doi: 10.1038/s41419-018-0937-7

Figure Lengend Snippet: Rescue experiments revealed that DSCR8 negatively regulated the miR-485-5p expression ( a , b ), whereas positively regulated both the mRNA ( a , b ) and protein ( c , d ) expression of FZD7 by DSCR8/miR-485-5p/FZD7 axis. c , d Western blot results from rescue experiments showed that DSCR8 positively regulated the accumulation of cytoplasmic β-catenin and the accumulation of nuclear β-catenin, c-Myc expression, and cyclin D1 expression by DSCR8/miR-485-5p/FZD7 axis. n = three repeats with similar results, * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: FZD7 Human cDNA ORF Clone (FZD7) and FZD7 siRNA (si-FZD7) were purchased from OriGene (OriGene Technologies,Inc., USA).

Techniques: Expressing, Western Blot

Journal: Cell Death & Disease

Article Title: Long non-coding RNA DSCR8 acts as a molecular sponge for miR-485-5p to activate Wnt/β-catenin signal pathway in hepatocellular carcinoma

doi: 10.1038/s41419-018-0937-7

Figure Lengend Snippet: DSCR8 acts as a molecular sponge for miR-485-5p to regulate FZD7 expression, subsequently activating Wnt/β-catenin signal pathway to promote HCC progression

Article Snippet: FZD7 Human cDNA ORF Clone (FZD7) and FZD7 siRNA (si-FZD7) were purchased from OriGene (OriGene Technologies,Inc., USA).

Techniques: Expressing

Journal: bioRxiv

Article Title: Cachd1 is a novel Frizzled- and LRP6-interacting protein required for neurons to acquire left-right asymmetric character

doi: 10.1101/2022.05.16.492129

Figure Lengend Snippet: (A, left) Representative scatter plot of flow cytometry testing binding of FLAG-tagged CACHD1 prey protein to human FZD7-eGFP transiently transfected HEK293E cells (detected by phycoerythrin (PE)-conjugated secondary antibody; A, right) without (blue) or with (red) pre-incubation with anti-Frizzled antibody OMP-18R5; secondary only negative control (grey). n = 3; one-tailed paired t -test (D. F. = 2, t = 9.53, *** p = 0.0054). (B) Dot plot of human (blue diamonds) or zebrafish (blue circles) CACHD1, or negative control CD200R (grey) prey protein binding (∆M PE ) to cells transiently transfected with eGFP fusion protein constructs indicated (transfections verified by antibody labelling in bold). Each dot represents a single experiment; horizontal bars denote the mean and error bars represent 95% confidence intervals. One way Welch test of means (Cachd1 prey v. CD200R prey, not assuming equal variances; F = 132.32, D. F num = 30.00, D. F denom = 34.67, p = 5.09 × 10 −28 ), post hoc pairwise t -tests with non-pooled standard deviations, Benjamini & Hochberg correction for multiple testing; only significant differences between Cachd1 and CD200R prey for individual transfections are presented here for clarity, ** 0.05 > p > 0.01, *** 0.01 > p > 0.005, **** p < 0.005. (C) SPR-based determination of K D for mouse CACHD1 ECD analyte binding to immobilised FZD5 CRD , LRP6 P3E3P4E4 (3-4, left panel), and normalised response curves for different CACHD1 ECD :FZD CRD interactions. RU, response units.

Article Snippet: Mouse Fzd5 cysteine rich domain (UniProt: Q9EQD0, residues A27-T157), human FZD7 CRD (UniProt: O75084, residues Q33-G170), human FZD8 CRD (UniProt: Q9H461, residues A28-T158) were synthesized (Genscript).

Techniques: Flow Cytometry, Binding Assay, Transfection, Incubation, Negative Control, One-tailed Test, Protein Binding, Construct

Journal: American Journal of Translational Research

Article Title: MicroRNA-206 attenuates glioma cell proliferation, migration, and invasion by blocking the WNT/β-catenin pathway via direct targeting of Frizzled 7 mRNA

doi:

Figure Lengend Snippet: Primer sequences for RT-qPCR

Article Snippet: J. FZD7 protein expression level was elevated in primary glioma patient samples as indicated by the Human Protein Atlas database (http://www.proteinatlas.org/) (The data shown are representative of three independent experiments, # P < 0.0001, ** P < 0.01, * P < 0.5).

Techniques: Sequencing

Journal: American Journal of Translational Research

Article Title: MicroRNA-206 attenuates glioma cell proliferation, migration, and invasion by blocking the WNT/β-catenin pathway via direct targeting of Frizzled 7 mRNA

doi:

Figure Lengend Snippet: MiR-206 directly targets FZD7 mRNA and inhibits the WNT pathway. A. The Venn diagram computationally predicting that miR-206 targets FZD7 mRNA according to five prediction algorithms: TargetScan, miRwalk, DIANAmT, miRanda, and PITA. B. After transfection with miR-ctrl or miR-206, the expression levels of FZD7, β-catenin, and c-Myc were determined by western blotting. Tubulin served as the loading control. C. The predicted miR-206-binding sequence in the FZD7 3’-UTR. D. Immunofluorescence staining of FZD7 (green) and β-catenin (red) in U87 and LN229 cells after transfection with miR-ctrl or miR-206. E, F. The expression levels of FZD7 in NBT and glioma samples were determined by western blotting. The fold changes were normalised to tubulin. NBT samples (n = 6) were collected from patients undergoing brain trauma surgery. Low-grade samples (n = 15) were derived from grade I and II gliomas, whereas high-grade samples (n = 18) were derived from grade III and IV gliomas. G. Pearson’s correlation analysis of the relative expression levels of miR-206 and relative protein levels of FZD7. H. MiR-206-binding sites in the 3’-UTR of FZD7 with wild-type (WT) or mutated (MUT) sequences highlighted. I. The dual-luciferase reporter assay was performed on U87 and LN229 cells, which were co-transfected with the indicated WT or mutant 3’-UTR construct and the miR-206 mimic. J. FZD7 protein expression level was elevated in primary glioma patient samples as indicated by the Human Protein Atlas database (http://www.proteinatlas.org/) (The data shown are representative of three independent experiments, #P < 0.0001, **P < 0.01, *P < 0.5).

Article Snippet: J. FZD7 protein expression level was elevated in primary glioma patient samples as indicated by the Human Protein Atlas database (http://www.proteinatlas.org/) (The data shown are representative of three independent experiments, # P < 0.0001, ** P < 0.01, * P < 0.5).

Techniques: Transfection, Expressing, Western Blot, Control, Binding Assay, Sequencing, Immunofluorescence, Staining, Derivative Assay, Luciferase, Reporter Assay, Mutagenesis, Construct

Journal: American Journal of Translational Research

Article Title: MicroRNA-206 attenuates glioma cell proliferation, migration, and invasion by blocking the WNT/β-catenin pathway via direct targeting of Frizzled 7 mRNA

doi:

Figure Lengend Snippet: Down-regulation of FZD7 had an effect similar to that of the over-expression of miR-206 in glioma cells. A. Western blotting analysis of FZD7, β-catenin, and c-Myc expression in U87 and LN229 cells after a knockdown of FZD7. B. The CCK-8 assay after cultivation for 96 h evaluated the proliferative ability of U87 and LN229 cells. C. Long-term cell viability was evaluated using a colony formation assay. D. Proliferation of cells was examined by the EdU incorporation assay. Representative images are shown (original magnification 200×). E. Migration of GBM cells was monitored using a wound-healing assay. (original magnification, 200×). F. Matrigel invasion assays uncovered the impact of the knockdown of FZD7 on GBM cell invasion. G, H. The cell cycle distribution of U87 and LN229 cells after a knockdown of FZD7 was analysed by flow cytometry. I. Migration of U87 and LN229 cells was monitored in a 3D spheroid migration assay. J. Invasion-associated proteins MMP9 and MMP2 in U87 and LN229 cells after a knockdown of FZD7 were quantified by western blotting. Tubulin served as the loading control. (Three independent experiments per group. Scale bar, 100 μm. ***P < 0.001).

Article Snippet: J. FZD7 protein expression level was elevated in primary glioma patient samples as indicated by the Human Protein Atlas database (http://www.proteinatlas.org/) (The data shown are representative of three independent experiments, # P < 0.0001, ** P < 0.01, * P < 0.5).

Techniques: Over Expression, Western Blot, Expressing, Knockdown, CCK-8 Assay, Colony Assay, Migration, Wound Healing Assay, Flow Cytometry, Control

Journal: American Journal of Translational Research

Article Title: MicroRNA-206 attenuates glioma cell proliferation, migration, and invasion by blocking the WNT/β-catenin pathway via direct targeting of Frizzled 7 mRNA

doi:

Figure Lengend Snippet: FZD7 re-introduction reverses the tumour-suppressive effect of miR-206 on GBM cells. A. A rescue experiment was conducted by introducing pcDNA3.1-FZD7 or pcDNA3.1 (in the presence or absence of ectopic miR-206 or miR-ctrl expression) into U87 and LN229 cells. Western blotting analysis of FZD7, β-catenin, and c-Myc in the indicated cells. Tubulin served as the loading control. B. The viability of glioma cells transfected with pcDNA3.1-FZD7 and miR-206 separately or together was evaluated in the CCK-8 assay. C. The cell proliferative potential was evaluated by the EdU assay, 48 h after co-transfection. D. A colony formation assay was performed to evaluate the long-term cell viability of GBM cells after co-transfection. E. Migration of GBM cells was monitored using a wound-healing assay. Respective images of GBM cells after co-transfection (original magnification, 200×). F. The cell cycle distribution of U87 and LN229 cells after co-transfection was analysed by flow cytometry. G, H. Matrigel invasion assays were performed to evaluate the invasive ability of GBM cells after co-transfection. I. 3D spheroid assays were performed on the indicated cells. J, K. Western blotting analysis illustrates the regulation of Invasion-associated proteins and EMT-associated proteins in the indicated cells. L. Western blotting analysis uncovered the effects on cell cycle-regulatory proteins in the indicated cells. (All experiments were performed in triplicate; ***P < 0.001, **P < 0.01, *P < 0.5).

Article Snippet: J. FZD7 protein expression level was elevated in primary glioma patient samples as indicated by the Human Protein Atlas database (http://www.proteinatlas.org/) (The data shown are representative of three independent experiments, # P < 0.0001, ** P < 0.01, * P < 0.5).

Techniques: Expressing, Western Blot, Control, Transfection, CCK-8 Assay, EdU Assay, Cotransfection, Colony Assay, Migration, Wound Healing Assay, Flow Cytometry

Journal: American Journal of Translational Research

Article Title: MicroRNA-206 attenuates glioma cell proliferation, migration, and invasion by blocking the WNT/β-catenin pathway via direct targeting of Frizzled 7 mRNA

doi:

Figure Lengend Snippet: MiR-206 attenuated tumour growth in a murine intracranial xenograft model. A. U87 cells pre-treated with a miR-ctrl-expressing or miR-206-expressing lentivirus (and a lentivirus expressing luciferase) were implanted into the brain of nude mice, and tumour formation was assessed by means of a non-invasive bioluminescence imaging system. Bioluminescent images were captured on days 0, 7, 14, 21, and 28 after the implantation. B. Overall survival was determined via Kaplan-Meier survival curves, and a log-rank test was conducted to assess the statistical significance of survival differences. C. After euthanasia, mouse brain tissues were collected, embedded into paraffin, and cut into thin sections for IHC analysis. The expression of FZD7, β-catenin, MMP2, and Ki-67 significantly diminished in the LV-miR-206 group.

Article Snippet: J. FZD7 protein expression level was elevated in primary glioma patient samples as indicated by the Human Protein Atlas database (http://www.proteinatlas.org/) (The data shown are representative of three independent experiments, # P < 0.0001, ** P < 0.01, * P < 0.5).

Techniques: Expressing, Luciferase, Imaging

Journal: Frontiers in Cellular Neuroscience

Article Title: Wingless-type family member 3A triggers neuronal polarization via cross-activation of the insulin-like growth factor-1 receptor pathway

doi: 10.3389/fncel.2013.00194

Figure Lengend Snippet: Activation with Wnt3a triggers the formation of a IFG-1r/Wnt3a/Fz7 complex. (A) Fz7 is enriched in GCPs. Western blot analysis of the distribution of the Wnt receptor Fz7 in different subcellular fractions of fetal brain: homogenate (H), low-speed supernatant (LSS), A-fraction (A), and growth cone particles (GCPs). Equal amounts of protein were loaded in all lines. Note the enrichment of Fz7 in GCPs around fourfold compared to (H). A blot showing the distribution of GAP-43, a protein highly enriched in GCPs, was included for comparison. (B , C) Wnt3a triggered the formation of a complex containing IGF-1r-Wnt3a-Fz7. Growth cone particles were incubated in control buffer or in the presence of 1.35 nM Wnt3a and immunoprecipitated with (B) Anti Wnt3a and probed with an antibody to the Wnt receptor Fz7 and (C) immunoprecipitated with β gc antibody to pull down IGF-1r and probed with anti Fz7. Immunoprecipitation of Wnt3a and β gc are shown to confirm successful IP and as a loading control. IP, immunoprecipitate; S, soluble fraction after immunoprecipitation. (D) Total protein from hippocampal neurons cultured for 20 h in the presence of 1.35 nm Wnt3a, deprived of any growth factor for 4 h, and stimulated (or not) with 1.35 nM Wnt3a for 5 min immunoprecipitated with anti β gc antibody to pull down the IGF-1r and probed with anti Fz7. Immunoprecipitation of β gc is shown to confirm successful IP and as a loading control. IP, immunoprecipitate; S, soluble fraction after immunoprecipitation. All blots are representative of at least three independent experiments.

Article Snippet: In order to characterize the direct interaction between IGF-1r and mouse Wnt3a or Fz7 [Recombinant Human FZD7/frizzled-7 Protein, made in HEK293 (aa 45–169-Speed Biosystems, MD, USA) preincubated with Wnt3a] IGF-1r was immobilized on a CM5 sensor chip surface, as described above.

Techniques: Activation Assay, Western Blot, Comparison, Incubation, Control, Immunoprecipitation, Cell Culture

Journal: Frontiers in Cellular Neuroscience

Article Title: Wingless-type family member 3A triggers neuronal polarization via cross-activation of the insulin-like growth factor-1 receptor pathway

doi: 10.3389/fncel.2013.00194

Figure Lengend Snippet: Wnt3a polarizing effects required normal expression of IGF-1r. (A) Triple immunofluorescence micrographs of hippocampal neurons after 24 h in culture showing the distribution of the IGF-1r β gc subunit, a scrambled sequence RNA (GFP, top) or IGF-1r targeted shRNA (GFP, bottom), and the neuronal marker β-III tubulin (tub). Cells were cultured in medium containing 1.35 nM Wnt3a. The ssRNA transfected cells (top) showed a normal morphology. In contrast, the shRNA transfected cells (bottom) did not develop an axon. (B) Western blots showing protein levels of β gc, the Wnt receptor Fz7 (to control specificity, also serves as loading control) and cells transfected with a scrambled RNA sequence (ssRNA), and cells transfected with IGF-1r shRNA (shRNA). We observed a substantial decrease in β gc expression in the shRNA treated cells. (C) Percentage (±sem) of neurons at different specific stages of differentiation grown for 20 h in culture in control medium or in the presence of 1.35 nM Wnt3a and transfected with a scrambled sequence RNA (ssRNA) or IGF-1r targeted shRNA (shRNA) ( n = 3 independent experiments). At least 100 transfected cells (GFP positive) were scored for each condition. * p < 0.005 compared to ssRNA + Wnt3a. Scale bar: 20 μm. (D) Immunofluorescence of hippocampal neurons after 24 h of DIV showing the distribution of myc (marker of transfection with cDNA encoding for a constitutively active form of the PI3k catalytic subunit p110). GFP is a marker of transfection efficiency with the shRNA directed to IGF-1r.Calibration bar = 20 μm. (E) Percentage (±sem) of neurons at different specific stages of differentiation grown for 20 h in culture in the presence of 1.35 nM Wnt3a and transfected with IGF-1r shRNA (shRNA) or co-transfected with shRNA and a constitutively active form of the PI3k catalytic subunit p110 (p110CA). n = 3 independent experiments. Thirty or more transfected or co-transfected cells were scored in each experiment. * p < 0.02 compared to shRNA.

Article Snippet: In order to characterize the direct interaction between IGF-1r and mouse Wnt3a or Fz7 [Recombinant Human FZD7/frizzled-7 Protein, made in HEK293 (aa 45–169-Speed Biosystems, MD, USA) preincubated with Wnt3a] IGF-1r was immobilized on a CM5 sensor chip surface, as described above.

Techniques: Expressing, Immunofluorescence, Sequencing, shRNA, Marker, Cell Culture, Transfection, Western Blot, Control

Journal: Frontiers in Cellular Neuroscience

Article Title: Wingless-type family member 3A triggers neuronal polarization via cross-activation of the insulin-like growth factor-1 receptor pathway

doi: 10.3389/fncel.2013.00194

Figure Lengend Snippet: (A) Wnt3a polarizing effect depends on the activation of Fz7. Double immunofluorescence micrographs of hippocampal neurons incubated for 24 h in the presence of 1.35 nM Wnt3a showing the distribution of the axonal marker Tau-1 and the neuronal marker β-III-tubulin. myc is a marker of transfection with the negative dominant form of Fz7-CRD. Scale bar: 20 mm. (B) Percentage (±sem) of neurons at different stages of differentiation grown for 20 h in medium containing 1.35 nM Wnt3a and transfected with pcDNA3.1-myc or Fz7-CRD ( n = 3 independent experiments). At least 20 transfected cells were scored for each condition. * p < 0.01 compared with pcDNA3.1-myc.

Article Snippet: In order to characterize the direct interaction between IGF-1r and mouse Wnt3a or Fz7 [Recombinant Human FZD7/frizzled-7 Protein, made in HEK293 (aa 45–169-Speed Biosystems, MD, USA) preincubated with Wnt3a] IGF-1r was immobilized on a CM5 sensor chip surface, as described above.

Techniques: Activation Assay, Immunofluorescence, Incubation, Marker, Transfection

Journal: Frontiers in Cellular Neuroscience

Article Title: Wingless-type family member 3A triggers neuronal polarization via cross-activation of the insulin-like growth factor-1 receptor pathway

doi: 10.3389/fncel.2013.00194

Figure Lengend Snippet: Wnt3a binds to Fz7 and cross-activate the IGF-1-PI3k-Akt pathway inducing initial axonal outgrowth and neuronal polarization. Challenging with 1.35 nM Wnt3a resulted in a polarized distribution of active (phosphorylated) IGF-1r and phosphorylated p85, the PI3k regulatory subunit, to one neurite of cells in stage 2 (see Figure ).

Article Snippet: In order to characterize the direct interaction between IGF-1r and mouse Wnt3a or Fz7 [Recombinant Human FZD7/frizzled-7 Protein, made in HEK293 (aa 45–169-Speed Biosystems, MD, USA) preincubated with Wnt3a] IGF-1r was immobilized on a CM5 sensor chip surface, as described above.

Techniques:

Journal: Nano research

Article Title: Inhibition of Wnt signaling by Frizzled7 antibody-coated nanoshells sensitizes triple-negative breast cancer cells to the autophagy regulator chloroquine

doi: 10.1007/s12274-020-2795-8

Figure Lengend Snippet: Depiction of the Wnt and autophagy pathways in TNBC. (a) Wnt signaling is activated in TNBC cells when Wnt ligands bind FZD7 cell surface receptors. This leads to nuclear translocation of β-catenin, which activates Wnt target genes to promote cell stemness and migration. (b) FZD7-NS suppress Wnt signaling in TNBC cells by blocking ligand/receptor interactions. When Wnt signaling is suppressed, autophagy is activated, contributing to cellular resistance. Applying the autophagy inhibitor CQ to TNBC cells in combination with FZD7-NS can overcome this resistance to impair aggressive cell behaviors.

Article Snippet: To conjugate either rabbit anti-human FZD7 (Fisher Scientific, or LifeSpan Biosciences, Seattle, WA, USA) or rabbit anti-human IgG control (Genscript, Piscataway, NJ, USA) antibodies to NS, anti-FZD7 or anti-IgG were linked to 5 kDa orthopyridyl disulfidepoly(ethylene glycol)-succinimidyl valerate (OPSS-PEG 5k -SVA) (Laysan Bio, Arab, AL, USA) linkers in sodium bicarbonate.

Techniques: Translocation Assay, Migration, Blocking Assay

Journal: Nano research

Article Title: Inhibition of Wnt signaling by Frizzled7 antibody-coated nanoshells sensitizes triple-negative breast cancer cells to the autophagy regulator chloroquine

doi: 10.1007/s12274-020-2795-8

Figure Lengend Snippet: Characterization of antibody-nanoshell (Ab-NS) conjugates. (a) Scheme depicting the synthesis of Ab-NS. (b) Extinction spectra of bare nanoshells versus nanoshells coated with IgG or FZD7 antibodies. (c) Hydrodynamic diameter (black) and zeta potential (blue) of bare NS, IgG-NS, and FZD7-NS. (d) SEM image of FZD7-NS. (e) Quantification of antibody loading on IgG-NS and FZD7-NS.

Article Snippet: To conjugate either rabbit anti-human FZD7 (Fisher Scientific, or LifeSpan Biosciences, Seattle, WA, USA) or rabbit anti-human IgG control (Genscript, Piscataway, NJ, USA) antibodies to NS, anti-FZD7 or anti-IgG were linked to 5 kDa orthopyridyl disulfidepoly(ethylene glycol)-succinimidyl valerate (OPSS-PEG 5k -SVA) (Laysan Bio, Arab, AL, USA) linkers in sodium bicarbonate.

Techniques: Zeta Potential Analyzer

Journal: Nano research

Article Title: Inhibition of Wnt signaling by Frizzled7 antibody-coated nanoshells sensitizes triple-negative breast cancer cells to the autophagy regulator chloroquine

doi: 10.1007/s12274-020-2795-8

Figure Lengend Snippet: Analysis of the intracellular trafficking of Cy5-labeled FZD7-NS. (a) Fluorescence microscopy images showing overlap of Cy5-labeled FZD7-NS (red) with various subcellular compartments (green) 24 h after the nanoparticles were added to the cell culture medium. Scale bars = 20 μm. (b) Results from quantitative colocalization analysis to calculate the fractional overlap of red-fluorescent FZD7-NS with green-fluorescent subcellular compartment and vice versa. MCC = Manders’ colocalization coefficient; letters a–d indicate grouping by one-way ANOVA with post hoc Turkey. Groups with different letters are significantly different at the 99% confidence level, except for the comparison of Rab8a and Rab 11 for MCC2, which has p = 0.026.

Article Snippet: To conjugate either rabbit anti-human FZD7 (Fisher Scientific, or LifeSpan Biosciences, Seattle, WA, USA) or rabbit anti-human IgG control (Genscript, Piscataway, NJ, USA) antibodies to NS, anti-FZD7 or anti-IgG were linked to 5 kDa orthopyridyl disulfidepoly(ethylene glycol)-succinimidyl valerate (OPSS-PEG 5k -SVA) (Laysan Bio, Arab, AL, USA) linkers in sodium bicarbonate.

Techniques: Labeling, Fluorescence, Microscopy, Cell Culture, Comparison

Journal: Nano research

Article Title: Inhibition of Wnt signaling by Frizzled7 antibody-coated nanoshells sensitizes triple-negative breast cancer cells to the autophagy regulator chloroquine

doi: 10.1007/s12274-020-2795-8

Figure Lengend Snippet: Examination of Wnt signaling and autophagy markers in TNBC cells exposed to various treatments. (a) and (b) qRT-PCR analysis of Axin2 and Cyclin D1 mRNA expression in MDA-MB-231 cells following treatment with media only (non-treated, NT), IgG-NS ± CQ, free FZD7 antibodies ± CQ, or FZD7-NS ± CQ. * p < 0.05 versus NT by one-way ANOVA with post hoc Turkey. # p < 0.05 by one-way ANOVA with post hoc Turkey. Precise p-values for all significant comparisons are provided in Table S2 in the ESM. (c) Western blot analysis of LC3 protein expression in MDA-MB-231 cells exposed to the autophagy promoter rapamycin, no treatment (NT), FZD7-NS, IgG-NS, or free FZD7 antibodies.

Article Snippet: To conjugate either rabbit anti-human FZD7 (Fisher Scientific, or LifeSpan Biosciences, Seattle, WA, USA) or rabbit anti-human IgG control (Genscript, Piscataway, NJ, USA) antibodies to NS, anti-FZD7 or anti-IgG were linked to 5 kDa orthopyridyl disulfidepoly(ethylene glycol)-succinimidyl valerate (OPSS-PEG 5k -SVA) (Laysan Bio, Arab, AL, USA) linkers in sodium bicarbonate.

Techniques: Quantitative RT-PCR, Expressing, Western Blot

Journal: Nano research

Article Title: Inhibition of Wnt signaling by Frizzled7 antibody-coated nanoshells sensitizes triple-negative breast cancer cells to the autophagy regulator chloroquine

doi: 10.1007/s12274-020-2795-8

Figure Lengend Snippet: Evaluation of TNBC cell migration in response to Wnt and autophagy inhibition. (a) Scheme depicting the transwell migration assay. (b) Representative fluorescence images of migrated GFP-expressing MDA-MB-231 cells 5 d after they were seeded in the transwell apparatus following treatment with NT, IgG-NS ± CQ, free FZD7 ± CQ, or FZD7-NS ± CQ. Scale = 1,000 μm. (c) Quantitative analysis of the number of migrated cells in each treatment group. # p < 0.01 versus FZD7-NS + CQ by one way ANOVA with post hoc Turkey. * p < 0.01 versus NT by one-way ANOVA with post hoc Turkey.

Article Snippet: To conjugate either rabbit anti-human FZD7 (Fisher Scientific, or LifeSpan Biosciences, Seattle, WA, USA) or rabbit anti-human IgG control (Genscript, Piscataway, NJ, USA) antibodies to NS, anti-FZD7 or anti-IgG were linked to 5 kDa orthopyridyl disulfidepoly(ethylene glycol)-succinimidyl valerate (OPSS-PEG 5k -SVA) (Laysan Bio, Arab, AL, USA) linkers in sodium bicarbonate.

Techniques: Migration, Inhibition, Transwell Migration Assay, Fluorescence, Expressing

Journal: Nano research

Article Title: Inhibition of Wnt signaling by Frizzled7 antibody-coated nanoshells sensitizes triple-negative breast cancer cells to the autophagy regulator chloroquine

doi: 10.1007/s12274-020-2795-8

Figure Lengend Snippet: Analysis of mRNA expression of several genes in MDA-MB-231 cells treated with FZD7-NS + CQ or controls. GAPDH was used as a housekeeping gene, and expression in each treatment group was normalized to that in the NT group. Data are means ± standard deviations. * indicates p < 0.05 versus NT and # indicates p < 0.05 versus FZD7-NS + CQ by one-way ANOVA with post hoc Turkey. Other significant differences are not shown for simplicity. Precise p-values for all significant comparisons are provided in Table S3 in the ESM.

Article Snippet: To conjugate either rabbit anti-human FZD7 (Fisher Scientific, or LifeSpan Biosciences, Seattle, WA, USA) or rabbit anti-human IgG control (Genscript, Piscataway, NJ, USA) antibodies to NS, anti-FZD7 or anti-IgG were linked to 5 kDa orthopyridyl disulfidepoly(ethylene glycol)-succinimidyl valerate (OPSS-PEG 5k -SVA) (Laysan Bio, Arab, AL, USA) linkers in sodium bicarbonate.

Techniques: Expressing

Journal: Nano research

Article Title: Inhibition of Wnt signaling by Frizzled7 antibody-coated nanoshells sensitizes triple-negative breast cancer cells to the autophagy regulator chloroquine

doi: 10.1007/s12274-020-2795-8

Figure Lengend Snippet: Self-renewal capacity of MDA-MB-231 cells subjected to Wnt and autophagy inhibition. (a) Scheme depicting the spheroid culture model; red cells illustrate breast cancer stem cells. (b) Representative bright-field images of spheroids cultured from MDA-MB-231 cells after exposure to NT, IgG-NS ± CQ, free FZD7 ± CQ, or FZD7-NS ± CQ. Scale bars = 50 μm. (c) Quantitative analysis of the number of spheroids with > 4 cells in each treatment group. *p < 0.01 compared to NT, while # p < 0.05 and ## p < 0.01 compared to FZD7-NS + CQ by one-way ANOVA with post hoc Turkey. Other comparisons are not depicted for simplicity.

Article Snippet: To conjugate either rabbit anti-human FZD7 (Fisher Scientific, or LifeSpan Biosciences, Seattle, WA, USA) or rabbit anti-human IgG control (Genscript, Piscataway, NJ, USA) antibodies to NS, anti-FZD7 or anti-IgG were linked to 5 kDa orthopyridyl disulfidepoly(ethylene glycol)-succinimidyl valerate (OPSS-PEG 5k -SVA) (Laysan Bio, Arab, AL, USA) linkers in sodium bicarbonate.

Techniques: Inhibition, Cell Culture