Journal: Research

Article Title: Isovalerylspiramycin I Reprograms the Immunosuppressive and Temozolomide-Resistant Microenvironment by Inhibiting the Frizzled-5/Wnt/β-Catenin Pathway in Glioblastoma

doi: 10.34133/research.0828

Figure Lengend Snippet: ISP-I targets FZD5 to inhibit the Wnt/β-catenin signaling pathway. (A) RNA-seq analysis indicated that ISP-I alters the transcriptome of LN-18. Volcano plot depicting the distribution and top 10 differentially expressed genes. (B) KEGG pathway enrichment analysis revealed classification and functional annotation of enriched pathways. The horizontal axis represented the number of genes, and the vertical axis showed the classification entries. (C) KEGG analysis highlighted significant suppression of Wnt/β-catenin signaling among altered pathways. (D) Normalized transcript expression values (nTPM) for CTNNB1 were calculated in GBM cell lines from the Human Protein Atlas project. (E) Changes in β-catenin levels were determined through Western blotting. (F) qRT-PCR analysis of CCND1 and Myc mRNA levels under ISP-I treatment ( n = 3). (G) Protein levels of cyclin D1 and c-Myc were measured by Western blotting. (H) The effect of ISP-I on levels of core proteins within the Wnt/β-catenin signaling pathway was measured by Western blotting in LN-18 cells. (I) The result of the molecular docking of ISP-I with FZD5 using Autodock Vina was visualized and simulated by PyMOL software. Interaction mode simulation of ISP-I and FZD5 (FZD5: green, ISP-I: yellow, interface residues: pink). (J) ISP-I increased the FZD5 thermal stability as assessed by temperature-dependent CETSA ( n = 3). (K) SPR assays were performed to quantify the binding affinity of ISP-I to FZD5. (L) Confocal microscopy images of FZD5 (red) and fluorescein-hydrazide-labeled ISP-I (green). Pearson’s coefficient was calculated from scatterplots (ImageJ). Scale bars = 10 μm. Data: mean ± SD; significance was calculated with one-way ANOVA test. ** P < 0.01; *** P < 0.001.

Article Snippet: FZD5 protein (10 μg/ml, 10473-H08H, Sino Bio, Beijing, China) was loaded at a flow rate (50 μl/min) for 60 s, followed by a dissociation flow period of 30 s at 25 °C.

Techniques: RNA Sequencing, Functional Assay, Expressing, Western Blot, Quantitative RT-PCR, Software, Binding Assay, Confocal Microscopy, Labeling

Journal: Research

Article Title: Isovalerylspiramycin I Reprograms the Immunosuppressive and Temozolomide-Resistant Microenvironment by Inhibiting the Frizzled-5/Wnt/β-Catenin Pathway in Glioblastoma

doi: 10.34133/research.0828

Figure Lengend Snippet: ISP-I targets FZD5, thereby inhibiting the Wnt/β-catenin signaling pathway. This inhibition leads to the suppression of PD-L1 and MGMT expression, augmenting glioblastoma cell susceptibility to TMZ and reprogramming the immunosuppressive microenvironment.

Article Snippet: FZD5 protein (10 μg/ml, 10473-H08H, Sino Bio, Beijing, China) was loaded at a flow rate (50 μl/min) for 60 s, followed by a dissociation flow period of 30 s at 25 °C.

Techniques: Inhibition, Expressing

Journal: STAR Protocols

Article Title: Protocol for changing gene expression in 3T3-L1 (pre)adipocytes using siRNA-mediated knockdown

doi: 10.1016/j.xpro.2024.103075

Figure Lengend Snippet:

Article Snippet: siFzd5, rGrCrArCrUrArArGrArCrGrGrArCrArArGrCrUrArGrArGAA , OriGene Technologies GmbH , SR417789, siRNA C.

Techniques: Recombinant, Modification, Reverse Transcription, Transfection, Random Hexamer Labeling, Software, Cell Culture, Electroporation, Inverted Microscopy

Journal: Angiogenesis

Article Title: Frizzled-5: A High Affinity Receptor for Secreted Frizzled-Related Protein-2 Activation of Nuclear Factor of Activated T-Cells c3 Signaling to Promote Angiogenesis

doi: 10.1007/s10456-017-9574-5

Figure Lengend Snippet: A) Full length homology model of SFRP2 and FZD5. CRD, the cysteine rich domain of FZD5, is colored green. TM, the transmembrane domain of FZD5, is colored orange along with the intracellular domains. B, C) Detail of twelve intermolecular interactions between SFRP2 and the CRD of FZD5.

Article Snippet: Gain and loss of function studies with FZD5 Stable transfection of 2H11 endothelial cells with FZD5 shRNA To silence FZD5 in endothelial cells, we used HuSH shRNA plasmids containing FZD5 (OriGene, Rockville MD, USA).

Techniques:

Journal: Angiogenesis

Article Title: Frizzled-5: A High Affinity Receptor for Secreted Frizzled-Related Protein-2 Activation of Nuclear Factor of Activated T-Cells c3 Signaling to Promote Angiogenesis

doi: 10.1007/s10456-017-9574-5

Figure Lengend Snippet: Fluorescence confocal microscopy was used to detect localization of SFRP2 (A, E, I) and FZD5 (B, F, J) in 2H11 (A–H) and SVR (I–L) cells. 2H11 cells were either untreated (A–D) or treated with 10 nM SFRP2 (E–H) for 1 minute. SFRP2 (green) FZD5 (red) were visualized using an FITC- or AlexaFluor546-labeled secondary antibody, respectively. Nuclei (blue) were identified using Hoeschst 33342. Merged images (C, G, K) showing the co-localization of SFRP2 and FZD5 (yellow) at the surface of SFRP2-treated 2H11 cells (G), at the surface SVR cells which highly express endogenous SFP2 (K), but not at the surface of untreated 2H11 cells (C). Controls were treated with secondary antibody only, with the primary antibody omitted (D, H, I). Scale bar in A, E and I represents 30μm.

Article Snippet: Gain and loss of function studies with FZD5 Stable transfection of 2H11 endothelial cells with FZD5 shRNA To silence FZD5 in endothelial cells, we used HuSH shRNA plasmids containing FZD5 (OriGene, Rockville MD, USA).

Techniques: Fluorescence, Confocal Microscopy, Labeling

Journal: Angiogenesis

Article Title: Frizzled-5: A High Affinity Receptor for Secreted Frizzled-Related Protein-2 Activation of Nuclear Factor of Activated T-Cells c3 Signaling to Promote Angiogenesis

doi: 10.1007/s10456-017-9574-5

Figure Lengend Snippet: The vertical scatter plot shows individual Pearson’s correlation coefficients with the mean of the coefficients and standard error of the mean (n = 9 cells from at least four fields for all samples). Analysis indicates that in 2H11 cells, a pool of SFRP2 and FZD5 colocalize. This phenomenon is rapidly amplified by the addition of exogenous SFRP2 and reaches the levels of colocalization observed in SVR cells, which express sustained endogenous levels of SFRP2. Difference between 2H11 control and 2H11 exposed for one minute to exogenous rhSFPR2 was significant (two tailed t-test; p = 0.0003).

Article Snippet: Gain and loss of function studies with FZD5 Stable transfection of 2H11 endothelial cells with FZD5 shRNA To silence FZD5 in endothelial cells, we used HuSH shRNA plasmids containing FZD5 (OriGene, Rockville MD, USA).

Techniques: Amplification, Two Tailed Test

Journal: Angiogenesis

Article Title: Frizzled-5: A High Affinity Receptor for Secreted Frizzled-Related Protein-2 Activation of Nuclear Factor of Activated T-Cells c3 Signaling to Promote Angiogenesis

doi: 10.1007/s10456-017-9574-5

Figure Lengend Snippet: A) Western blot analysis on samples from sham (control) and shFZD5-transfected 2H11 cells treated for 0, 5 and 15 min with 10nM SFRP2, showing the levels of SFRP2 and FZD5. Actin was used as a loading control. Dosimetry units (DU) normalized to actin. B) Sham and shFZD5 samples from 2H11 cells treated for 0, 5 and 15 with 10nM SFRP2 were immunoprecipitated with an anti-SFRP2 antibody (lanes 1 to 6) or a control IgG (lanes 7 to 12) and the levels of SFRP2 and FZD5 were then measured by western blot. C) Western blot analysis on samples from GFP (control) and FZD5/GFP expressing MS1 cells treated for 0, 5 and 15 min with 10nM SFRP2, showing the levels of SFRP2 and FZD5. Actin was used as a loading control, and DU are normalized to actin. D) GFP (control) and FZD5/GFP samples from MS1 cells treated for 0, 5 and 15 with 10nM SFRP2 were immunoprecipitated with an anti-SFRP2 antibody (lanes 1 to 6) or a control IgG (lanes 7 to 12) and the levels of SFRP2 and FZD5 were then measured by western blot.

Article Snippet: Gain and loss of function studies with FZD5 Stable transfection of 2H11 endothelial cells with FZD5 shRNA To silence FZD5 in endothelial cells, we used HuSH shRNA plasmids containing FZD5 (OriGene, Rockville MD, USA).

Techniques: Western Blot, Transfection, Immunoprecipitation, Expressing

Journal: Angiogenesis

Article Title: Frizzled-5: A High Affinity Receptor for Secreted Frizzled-Related Protein-2 Activation of Nuclear Factor of Activated T-Cells c3 Signaling to Promote Angiogenesis

doi: 10.1007/s10456-017-9574-5

Figure Lengend Snippet: Graphic representation of rhSFRP2 and rhWnt5a binding to FZD5-Fc showing changes in 450 nm optical density (OD450) as a function of the log of ligand concentrations. To do this, A microplate solid phase protein binding assay was performed using a concentration range of his-tagged recombinant human SFRP2 and Wnt5a from 24nM – 1500nM (x axis). The experiment was repeated twice for a total of n = 6. Kd, EC50 and Hill coefficient were calculated and compared for SFRP2 and Wnt5a binding to FZD5.

Article Snippet: Gain and loss of function studies with FZD5 Stable transfection of 2H11 endothelial cells with FZD5 shRNA To silence FZD5 in endothelial cells, we used HuSH shRNA plasmids containing FZD5 (OriGene, Rockville MD, USA).

Techniques: Binding Assay, Protein Binding, Concentration Assay, Recombinant

Journal: Angiogenesis

Article Title: Frizzled-5: A High Affinity Receptor for Secreted Frizzled-Related Protein-2 Activation of Nuclear Factor of Activated T-Cells c3 Signaling to Promote Angiogenesis

doi: 10.1007/s10456-017-9574-5

Figure Lengend Snippet: A, B) Bar graphs showing the number of branch points (± SEM) in tube formation assays using 2H11 (A) and MS1 cells (B). Sham (control) and shFZD5-transfected 2H11 cells (A), or GFP (control) and FZD5/GFP-expressing MS1 cells (B) were plated in a Matrigel® tube formation assay and treated with either control media or SFRP2 10nM for 4 hours. Experiments were repeated 3 times (n = 12 total). C, D) Bar graphs showing the number of branch points (± SEM) in tube formation assays using 2H11 (C) and HMEC-1 endothelial cells (D). Cells were plated in a tube formation assay and treated with either control media, SFRP2 30nM, FZD5-FC 500 ng/ml, or SFRP2 30nM + FD5-FC 500 ng/ml for 4 hours. Experiments were repeated 3 times (n=12 for HMEC-1 and n=9 for 2H11). E,F) Bar graphs showing calcium levels measured (RFU ± SEM) in sham and shFZD5-transfected 2H11 cells depending on the concentrations of SFRP2 (E; 1–30nM) or Wnt5a (F; 0.7–20nM). Each experiment was repeated 4 times (n = 16). G) Western blot showing the levels of nuclear NFATc3 in sham (control) and shFZD5-transfected 2H11 cells treated with recombinant media or mouse recombinant SFRP2 (10nM) for 1 hour. TATA was used as an internal control, and DU (dosimetry units) normalized to TATA. *: p-value<0.05.

Article Snippet: Gain and loss of function studies with FZD5 Stable transfection of 2H11 endothelial cells with FZD5 shRNA To silence FZD5 in endothelial cells, we used HuSH shRNA plasmids containing FZD5 (OriGene, Rockville MD, USA).

Techniques: Transfection, Expressing, Tube Formation Assay, Western Blot, Recombinant

Journal: Angiogenesis

Article Title: Frizzled-5: A High Affinity Receptor for Secreted Frizzled-Related Protein-2 Activation of Nuclear Factor of Activated T-Cells c3 Signaling to Promote Angiogenesis

doi: 10.1007/s10456-017-9574-5

Figure Lengend Snippet: A) Full length homology model of SFRP2 and FZD5. CRD, the cysteine rich domain of FZD5, is colored green. TM, the transmembrane domain of FZD5, is colored orange along with the intracellular domains. B, C) Detail of twelve intermolecular interactions between SFRP2 and the CRD of FZD5.

Article Snippet: Stable transfection of 2H11 endothelial cells with FZD5 shRNA To silence FZD5 in endothelial cells, we used HuSH shRNA plasmids containing FZD5 (OriGene, Rockville MD, USA).

Techniques:

Journal: Angiogenesis

Article Title: Frizzled-5: A High Affinity Receptor for Secreted Frizzled-Related Protein-2 Activation of Nuclear Factor of Activated T-Cells c3 Signaling to Promote Angiogenesis

doi: 10.1007/s10456-017-9574-5

Figure Lengend Snippet: Fluorescence confocal microscopy was used to detect localization of SFRP2 (A, E, I) and FZD5 (B, F, J) in 2H11 (A–H) and SVR (I–L) cells. 2H11 cells were either untreated (A–D) or treated with 10 nM SFRP2 (E–H) for 1 minute. SFRP2 (green) FZD5 (red) were visualized using an FITC- or AlexaFluor546-labeled secondary antibody, respectively. Nuclei (blue) were identified using Hoeschst 33342. Merged images (C, G, K) showing the co-localization of SFRP2 and FZD5 (yellow) at the surface of SFRP2-treated 2H11 cells (G), at the surface SVR cells which highly express endogenous SFP2 (K), but not at the surface of untreated 2H11 cells (C). Controls were treated with secondary antibody only, with the primary antibody omitted (D, H, I). Scale bar in A, E and I represents 30μm.

Article Snippet: Stable transfection of 2H11 endothelial cells with FZD5 shRNA To silence FZD5 in endothelial cells, we used HuSH shRNA plasmids containing FZD5 (OriGene, Rockville MD, USA).

Techniques: Fluorescence, Confocal Microscopy, Labeling

Journal: Angiogenesis

Article Title: Frizzled-5: A High Affinity Receptor for Secreted Frizzled-Related Protein-2 Activation of Nuclear Factor of Activated T-Cells c3 Signaling to Promote Angiogenesis

doi: 10.1007/s10456-017-9574-5

Figure Lengend Snippet: The vertical scatter plot shows individual Pearson’s correlation coefficients with the mean of the coefficients and standard error of the mean (n = 9 cells from at least four fields for all samples). Analysis indicates that in 2H11 cells, a pool of SFRP2 and FZD5 colocalize. This phenomenon is rapidly amplified by the addition of exogenous SFRP2 and reaches the levels of colocalization observed in SVR cells, which express sustained endogenous levels of SFRP2. Difference between 2H11 control and 2H11 exposed for one minute to exogenous rhSFPR2 was significant (two tailed t-test; p = 0.0003).

Article Snippet: Stable transfection of 2H11 endothelial cells with FZD5 shRNA To silence FZD5 in endothelial cells, we used HuSH shRNA plasmids containing FZD5 (OriGene, Rockville MD, USA).

Techniques: Amplification, Two Tailed Test

Journal: Angiogenesis

Article Title: Frizzled-5: A High Affinity Receptor for Secreted Frizzled-Related Protein-2 Activation of Nuclear Factor of Activated T-Cells c3 Signaling to Promote Angiogenesis

doi: 10.1007/s10456-017-9574-5

Figure Lengend Snippet: A) Western blot analysis on samples from sham (control) and shFZD5-transfected 2H11 cells treated for 0, 5 and 15 min with 10nM SFRP2, showing the levels of SFRP2 and FZD5. Actin was used as a loading control. Dosimetry units (DU) normalized to actin. B) Sham and shFZD5 samples from 2H11 cells treated for 0, 5 and 15 with 10nM SFRP2 were immunoprecipitated with an anti-SFRP2 antibody (lanes 1 to 6) or a control IgG (lanes 7 to 12) and the levels of SFRP2 and FZD5 were then measured by western blot. C) Western blot analysis on samples from GFP (control) and FZD5/GFP expressing MS1 cells treated for 0, 5 and 15 min with 10nM SFRP2, showing the levels of SFRP2 and FZD5. Actin was used as a loading control, and DU are normalized to actin. D) GFP (control) and FZD5/GFP samples from MS1 cells treated for 0, 5 and 15 with 10nM SFRP2 were immunoprecipitated with an anti-SFRP2 antibody (lanes 1 to 6) or a control IgG (lanes 7 to 12) and the levels of SFRP2 and FZD5 were then measured by western blot.

Article Snippet: Stable transfection of 2H11 endothelial cells with FZD5 shRNA To silence FZD5 in endothelial cells, we used HuSH shRNA plasmids containing FZD5 (OriGene, Rockville MD, USA).

Techniques: Western Blot, Transfection, Immunoprecipitation, Expressing

Journal: Angiogenesis

Article Title: Frizzled-5: A High Affinity Receptor for Secreted Frizzled-Related Protein-2 Activation of Nuclear Factor of Activated T-Cells c3 Signaling to Promote Angiogenesis

doi: 10.1007/s10456-017-9574-5

Figure Lengend Snippet: Graphic representation of rhSFRP2 and rhWnt5a binding to FZD5-Fc showing changes in 450 nm optical density (OD450) as a function of the log of ligand concentrations. To do this, A microplate solid phase protein binding assay was performed using a concentration range of his-tagged recombinant human SFRP2 and Wnt5a from 24nM – 1500nM (x axis). The experiment was repeated twice for a total of n = 6. Kd, EC50 and Hill coefficient were calculated and compared for SFRP2 and Wnt5a binding to FZD5.

Article Snippet: Stable transfection of 2H11 endothelial cells with FZD5 shRNA To silence FZD5 in endothelial cells, we used HuSH shRNA plasmids containing FZD5 (OriGene, Rockville MD, USA).

Techniques: Binding Assay, Protein Binding, Concentration Assay, Recombinant

Journal: Angiogenesis

Article Title: Frizzled-5: A High Affinity Receptor for Secreted Frizzled-Related Protein-2 Activation of Nuclear Factor of Activated T-Cells c3 Signaling to Promote Angiogenesis

doi: 10.1007/s10456-017-9574-5

Figure Lengend Snippet: A, B) Bar graphs showing the number of branch points (± SEM) in tube formation assays using 2H11 (A) and MS1 cells (B). Sham (control) and shFZD5-transfected 2H11 cells (A), or GFP (control) and FZD5/GFP-expressing MS1 cells (B) were plated in a Matrigel® tube formation assay and treated with either control media or SFRP2 10nM for 4 hours. Experiments were repeated 3 times (n = 12 total). C, D) Bar graphs showing the number of branch points (± SEM) in tube formation assays using 2H11 (C) and HMEC-1 endothelial cells (D). Cells were plated in a tube formation assay and treated with either control media, SFRP2 30nM, FZD5-FC 500 ng/ml, or SFRP2 30nM + FD5-FC 500 ng/ml for 4 hours. Experiments were repeated 3 times (n=12 for HMEC-1 and n=9 for 2H11). E,F) Bar graphs showing calcium levels measured (RFU ± SEM) in sham and shFZD5-transfected 2H11 cells depending on the concentrations of SFRP2 (E; 1–30nM) or Wnt5a (F; 0.7–20nM). Each experiment was repeated 4 times (n = 16). G) Western blot showing the levels of nuclear NFATc3 in sham (control) and shFZD5-transfected 2H11 cells treated with recombinant media or mouse recombinant SFRP2 (10nM) for 1 hour. TATA was used as an internal control, and DU (dosimetry units) normalized to TATA. *: p-value<0.05.

Article Snippet: Stable transfection of 2H11 endothelial cells with FZD5 shRNA To silence FZD5 in endothelial cells, we used HuSH shRNA plasmids containing FZD5 (OriGene, Rockville MD, USA).

Techniques: Transfection, Expressing, Tube Formation Assay, Western Blot, Recombinant

Journal: International Journal of Clinical and Experimental Pathology

Article Title: miR-149 regulates the proliferation and apoptosis of human colonic carcinoma cells by targeting FZD5

doi:

Figure Lengend Snippet: FZD5 is a direct target of miR-149. SW480 cells were transfected with miR-149 mimics or miR-NC. A. Expression level of FZD5 detected by Q-PCR method. B, C. Expression level of FZD5 checked by western blot. D. Relative activities of luciferase reporters. Expression level of FZD5 detected by qRT-PCR analysis. Data are the mean ± SD; each bar represents the mean of three independent experiments carried out in triplicate. *P<0.05, compared with miR-NC group. **P<0.01, compared with miR-NC group.

Article Snippet: The human miR-149 duplex mimic and negative control oligonucleotide duplex mimic (miR-NC), small interfering RNA (siRNA) for Frizzled class receptor 5 (FZD5) and the negative control RNA (siRNA-NC) were designed and synthesized by Genepharma (Suzhou, China).

Techniques: Transfection, Expressing, Western Blot, Luciferase, Quantitative RT-PCR

Journal: International Journal of Clinical and Experimental Pathology

Article Title: miR-149 regulates the proliferation and apoptosis of human colonic carcinoma cells by targeting FZD5

doi:

Figure Lengend Snippet: miR-149 regulates cell apoptosis by targeting FZD5. A. miR-NC group. B. miR-149 mimics group. C. miR-149 mimics + SiFZD5 group. D. Quantitative analysis of cell apoptosis in CRC cells of each group. Data are the mean ± SD; each bar represents the mean of three independent experiments carried out in triplicate. **P<0.01, compared with miR-NC group. ##P<0.01, compared with the miR-mimics group.

Article Snippet: The human miR-149 duplex mimic and negative control oligonucleotide duplex mimic (miR-NC), small interfering RNA (siRNA) for Frizzled class receptor 5 (FZD5) and the negative control RNA (siRNA-NC) were designed and synthesized by Genepharma (Suzhou, China).

Techniques:

Journal: International Journal of Clinical and Experimental Pathology

Article Title: miR-149 regulates the proliferation and apoptosis of human colonic carcinoma cells by targeting FZD5

doi:

Figure Lengend Snippet: miR-149 inhibits activation of the Wnt/β-catenin signaling pathway. The expression of β-catenin and cyclinD1 were detected by western blot. Data are the mean ± SD; each bar represents the mean of three independent experiments carried out in triplicate. **P<0.01, compared with miR-NC group. ##P<0.01, compared with miR-mimics group.

Article Snippet: The human miR-149 duplex mimic and negative control oligonucleotide duplex mimic (miR-NC), small interfering RNA (siRNA) for Frizzled class receptor 5 (FZD5) and the negative control RNA (siRNA-NC) were designed and synthesized by Genepharma (Suzhou, China).

Techniques: Activation Assay, Expressing, Western Blot