Journal: Cell Reports Medicine

Article Title: Schwann cell-secreted frizzled-related protein 1 dictates neuroinflammation and peripheral nerve degeneration after neurotrauma

doi: 10.1016/j.xcrm.2024.101791

Figure Lengend Snippet: Figure 1. sFRP1 was abundantly produced in nerve ECM following injury and associated with nerve degeneration (A) The isolation of sciatic nerve samples and proteomic analysis process. (B) The clustering distribution of injured and uninjured nerve samples as plotted by PCA analysis. (C) Differentially expressed proteins between injured and uninjured nerve samples are displayed in volcano plot. N = 3 mice. Proteins regulated over 1.5-fold changes (adj. p < 0.05) are highlighted in blue (downregulated) and red (upregulated). (D) GO enrichment analysis indicating the classification of differentially expressed proteins related to the biological process category. (E) Differentially expressed proteins in the GO category of ECM are displayed as a heatmap. (F) Western blotting analysis demonstrating increased production of sFRP1 in the injured nerve tissue. (G) Quantification of sFRP1 protein level in sciatic nerves isolated from uninjured and injured mice as indicated by western blot analysis. N = 3 mice. (H) Representative TEM, HE, and TB images of injured nerves isolated from mice treated with WAY-316606 and PBS at 3 weeks post injury. N = 6 mice. (I and J) Quantification of myelinated axon diameter and g-ratio as indicated in TEM images. (K) Quantification of myelinated axon density as indicated in HE-stained images. (L) Representative IHC images of human nerves stained for sFRP1 at 12 h after injury. Statistical significance was determined using two-tailed unpaired Student’s t tests; **p < 0.01; *p < 0.05; ns, no difference. Data were presented as mean ± SD.

Article Snippet: For recombinant sFRP1 protein treatment, mice were intraneurally injected with sFRP1 protein (HY-P73413, MedChemExpress, 500nM, 5mL) or PBS (5mL), immediately after nerve transection procedure.

Techniques: Produced, Isolation, Western Blot, Staining, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Schwann cell-secreted frizzled-related protein 1 dictates neuroinflammation and peripheral nerve degeneration after neurotrauma

doi: 10.1016/j.xcrm.2024.101791

Figure Lengend Snippet: Figure 3. Mice with deletion of sFRP1 in SCs profoundly reduced macrophage infiltration and improved nerve regeneration (A) Sfrp1flox/flox mice were bred with PlpcreErt1 mice to generate tamoxifen-inducible SC-specific sFRP1 knockout (Sfrp1flox/floxPlpcreErt1) and littermate control (Sfrp1flox/flox) mice. (B and C) Representative SCG10 immunostaining and related quantification of sciatic nerves at 14 days post transection. N = 6 mice. The dashed line indicates the transection site. Scale bar, 500 mm. (D and E) Representative F4/80 immunostaining (red) of sciatic nerves taken from the injury site, 1,000, 2,000, and 3,000 mm distal to the injury site and related quantification of infiltrated macrophages. Scale bar, 100 mm. N = 6 mice. (F and G) Western blot analysis and related quantification of TNF-a level in injured nerves at 24 h post transection. N = 3 mice. (H and I) Triple staining of CCL2 (green), F4/80 (red), and NeuN (pink) on sciatic DRG sections from Sfrp1flox/flox and Sfrp1flox/floxPlpcreErt1 mice and related quantification of CCL expression level in DRGs. N = 6 mice. No significant difference of CCL2 expression is observed between DRGs of Sfrp1flox/flox and Sfrp1flox/floxPlpcreErt1 mice. (J–L) Representative TUBB3 immunostaining (green) of sciatic DRG neurons isolated from Sfrp1flox/flox and Sfrp1flox/floxPlpcreErt1 mice (n = 6 mice) and related quantification. DRG neurons were cultured in vitro for 4 days or 7 days. (M and N) Representative immunostaining and related quantification of ATF3 (red) and the neuronal marker NeuN (green) in sciatic DRGs at 24 h after nerve injury. N = 6 mice. Scale bar, 50 mm. Statistical significance in (C) and (E) was analyzed by two-way ANOVA followed by Sidak’s post hoc analysis. Statistical significance was determined using two- tailed unpaired Student’s t tests; ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05; ns, no significance. Data were presented as mean ± SD.

Article Snippet: For recombinant sFRP1 protein treatment, mice were intraneurally injected with sFRP1 protein (HY-P73413, MedChemExpress, 500nM, 5mL) or PBS (5mL), immediately after nerve transection procedure.

Techniques: Knock-Out, Control, Immunostaining, Western Blot, Staining, Expressing, Isolation, Cell Culture, In Vitro, Marker, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Schwann cell-secreted frizzled-related protein 1 dictates neuroinflammation and peripheral nerve degeneration after neurotrauma

doi: 10.1016/j.xcrm.2024.101791

Figure Lengend Snippet: Figure 4. SFRP1 induces the F4/80+ CD86+ proinflammatory macrophage phenotype and inhibits oxidative metabolism (A and B) The axon length of sciatic DRG neurons demonstrates no significant difference in response to sFRP1 treatment. N = 6 biological replicates. (C) Representative TEM images reveal that the morphology and structure of mitochondria were well preserved in sFRP1-treated neurons. (D and E) Representative TEM images and related quantification of nerve transections (N = 6 mice). The suppressing effect of sFRP1 on axon regrowth is alleviated in a macrophage-deficient condition. (F) Double staining of IL-1b (red) and TNF-a (green) on sFRP1-treated BMDMs. (G) sFRP1-induced phenotypic switch is revealed by flow cytometric quantification. FITC reflects F4/80-positive cells. PE reflects CD206-positive cells. APC reflects CD86-positive cells. (H and I) Quantification of the percentage of IL-1b and TNF-a-positive cells as reflected by Figure 4F. Biological replicates n = 3 with two technical replicates each. (J) Double staining of Arg-1 (red) and Wnt3a (green) on sFRP1 and PBS-treated BMDMs. (K) The internalizing capacity of BMDMs was measured by incubating with 100 mg/mL pHrodo BioParticles (green). BMDMs were visualized by F4/80 (red) staining.

Article Snippet: For recombinant sFRP1 protein treatment, mice were intraneurally injected with sFRP1 protein (HY-P73413, MedChemExpress, 500nM, 5mL) or PBS (5mL), immediately after nerve transection procedure.

Techniques: Double Staining, Staining

Journal: Cell Reports Medicine

Article Title: Schwann cell-secreted frizzled-related protein 1 dictates neuroinflammation and peripheral nerve degeneration after neurotrauma

doi: 10.1016/j.xcrm.2024.101791

Figure Lengend Snippet: Figure 6. Depletion of HSP90 in macrophages attenuated neuroinflammation and nerve degenerative changes exerted by sFRP1 (A) Hsp90aaflox/+ mice were bred with Lyz2-cre mice to generate macrophage-specific HSP90-deficient (Hsp90aaflox/+Lyz2-cre) and littermate control (Hsp90aaflox/+) mice. (B and C) Representative IF images of SCG10 staining and related quantification of sciatic nerves at 2 weeks post injury. The dashed line indicates the transection site. Scale bar, 500 mm. N = 6 mice. (D and E) Representative IF images of F4/80 staining (red) of sciatic nerves and related quantification of macrophages at 2 weeks post injury. Scale bar, 100 mm. N = 6 mice. (F–I) Double staining of TNF-a (red) and IL-1b (green) on nerve longitudinal sections and related quantification. (J–L) Representative TUBB3 staining (green) and related quantification of sciatic DRG neurons isolated from Hsp90aaflox/+ and Hsp90aaflox/+Lyz2-cre mice after 4 days and 7 days of culture. Biological replicates n = 3 with two technical replicates each. Statistical significance was determined using two-way ANOVA followed by Sidak’s post hoc analysis in (C) and (E), and using two-tailed unpaired Student’s t tests in (F), (G), (K), and (L); **p < 0.01; ***p < 0.001; *p < 0.05; ns, no significance. Data were presented as mean ± SD.

Article Snippet: For recombinant sFRP1 protein treatment, mice were intraneurally injected with sFRP1 protein (HY-P73413, MedChemExpress, 500nM, 5mL) or PBS (5mL), immediately after nerve transection procedure.

Techniques: Control, Staining, Double Staining, Isolation, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Schwann cell-secreted frizzled-related protein 1 dictates neuroinflammation and peripheral nerve degeneration after neurotrauma

doi: 10.1016/j.xcrm.2024.101791

Figure Lengend Snippet: Figure 7. SFRP1-neutralizing antibody treatment improved axon regeneration in vivo and in vitro (A and B) Representative SCG10 immunostaining and related quantification of murine injured nerves at 2 weeks after nerve transection. The dashed line indicates the transection site. Scale bar, 500 mm. N = 6 mice. (C) Schematic diagram of DRG neuron and macrophage microfluidic coculture chamber assay. (D) Representative optical images of macrophages in the neuron-macrophage coculture chambers. (E and F) Representative TUBB3 immunofluorescent images of neurons in the neuron-macrophage co-culture chambers and related quantification of average axon length in microfluidic channels. Biological replicates n = 3 with two technical replicates each. (G) Schematic diagram of DRG neuron and macrophage direct coculture assay. (H and I) Representative IF images stained for TUBB3 (green) on sciatic DRG neurons, and quantification of average axon length per cell in the direct coculture dishes. Biological replicates n = 3 with two technical replicates each. Statistical significance was determined using two-way ANOVA followed by Sidak’s post hoc analysis in (B) and (I) and using two-tailed unpaired Student’s t tests in (F); ***p < 0.001; **p < 0.01; *p < 0.05. Data were presented as mean ± SD.

Article Snippet: For recombinant sFRP1 protein treatment, mice were intraneurally injected with sFRP1 protein (HY-P73413, MedChemExpress, 500nM, 5mL) or PBS (5mL), immediately after nerve transection procedure.

Techniques: In Vivo, In Vitro, Immunostaining, Boyden Chamber Assay, Co-Culture Assay, Co-culture Assay, Staining, Two Tailed Test

Journal: Journal of Personalized Medicine

Article Title: Impact of REAC Regenerative Endogenous Bioelectrical Cell Reprogramming on MCF7 Breast Cancer Cells

doi: 10.3390/jpm13061019

Figure Lengend Snippet: DKK1 and SFRP1 quantification by ELISA. The concentrations of DKK1 ( A ) and SFRP1 ( B ) were measured after 7, 10, and 14 days in supernatants of MCF7 either exposed or not to REAC TO-RGN treatment. Data are expressed as mean ± SD relative to the control (mean ± SD) (** p ≤ 0.01; *** p ≤ 0.001).

Article Snippet: The concentrations of human secreted frizzled-related protein 1 (SFRP1) and human Dickkopf-related protein 1 (DKK1) were determined using the human secreted frizzled-related protein 1 (SFRP1) ELISA Kit (Cusabio, Flarebio Biotech LLC) and the human DKK1 (Dickkopf-related protein 1) ELISA Kit (Elabscience Biotechnology Co., Ltd, Houston, TX, USA), respectively.

Techniques: Enzyme-linked Immunosorbent Assay, Control

Journal: Cell Reports Medicine

Article Title: Schwann cell-secreted frizzled-related protein 1 dictates neuroinflammation and peripheral nerve degeneration after neurotrauma

doi: 10.1016/j.xcrm.2024.101791

Figure Lengend Snippet: sFRP1 was abundantly produced in nerve ECM following injury and associated with nerve degeneration (A) The isolation of sciatic nerve samples and proteomic analysis process. (B) The clustering distribution of injured and uninjured nerve samples as plotted by PCA analysis. (C) Differentially expressed proteins between injured and uninjured nerve samples are displayed in volcano plot. N = 3 mice. Proteins regulated over 1.5-fold changes (adj. p < 0.05) are highlighted in blue (downregulated) and red (upregulated). (D) GO enrichment analysis indicating the classification of differentially expressed proteins related to the biological process category. (E) Differentially expressed proteins in the GO category of ECM are displayed as a heatmap. (F) Western blotting analysis demonstrating increased production of sFRP1 in the injured nerve tissue. (G) Quantification of sFRP1 protein level in sciatic nerves isolated from uninjured and injured mice as indicated by western blot analysis. N = 3 mice. (H) Representative TEM, HE, and TB images of injured nerves isolated from mice treated with WAY-316606 and PBS at 3 weeks post injury. N = 6 mice. (I and J) Quantification of myelinated axon diameter and g-ratio as indicated in TEM images. (K) Quantification of myelinated axon density as indicated in HE-stained images. (L) Representative IHC images of human nerves stained for sFRP1 at 12 h after injury. Statistical significance was determined using two-tailed unpaired Student’s t tests; ∗∗ p < 0.01; ∗ p < 0.05; ns, no difference. Data were presented as mean ± SD.

Article Snippet: BMDMs were lysed in RIPA lysis buffer and incubated with His-labeled sFRP1 (Ag24844, Proteintech).

Techniques: Produced, Isolation, Western Blot, Staining, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Schwann cell-secreted frizzled-related protein 1 dictates neuroinflammation and peripheral nerve degeneration after neurotrauma

doi: 10.1016/j.xcrm.2024.101791

Figure Lengend Snippet: SCs sensed injury signals to release sFRP1 and elicited mesenchymal traits (A) t-distributed stochastic neighbor embedding (t-SNE) plot shows clustering of nerve cells based on gene expression. Single-cell sequencing datasets are analyzed from GSE120678 . BC, B cell; TC, T cell; EC, endothelial cell; Macro, macrophage; SC, Schwann cell; Endo, endoneurial fibroblast; Epi, epineurial fibroblast; Peri, perineurial fibroblast. (B) sFRP1 expression is mainly distributed in SCs and fibroblasts in both uninjured and injured sciatic nerves. (C and D) Double IF staining of S100β (red)/sFRP1 (green) and Fibro (red)/sFRP1 (green) on both longitudinal (C) and transverse (D) sections of sciatic nerves. (E and F) Percentage of sFRP1-positive SCs and fibroblasts in uninjured and injured nerves. N = 6 mice. (G) Illustration of the in vivo LPS treatment design. (H) sFRP1 protein level in SCs isolated from PBS or LPS (15 mg/kg) intraperitoneally treated mice. (I) Quantification of sFRP1 protein level in sciatic nerves isolated from LPS-treated and PBS-treated mice as indicated by western blot analysis. N = 3 biological replicates. (J) Illustration of the in vitro LPS treatment design. (K) Western blot analysis of sFRP1 protein level in SCs treated with different concentrations of LPS. (L and M) Quantification of fluorescence intensity of sFRP1 and PDGFRα staining in SCs. (N) sFRP1 (green) and PDGFRα (red) double staining on LPS-treated and PBS-treated SCs. N = 3 biological replicates. Two fields were quantified as technical replicates in each biological replicates. Statistical significance was determined using two-tailed unpaired Student’s t tests; ∗∗∗∗ p < 0.0001; ∗∗ p < 0.01; ∗ p < 0.05. Data were presented as mean ± SD.

Article Snippet: BMDMs were lysed in RIPA lysis buffer and incubated with His-labeled sFRP1 (Ag24844, Proteintech).

Techniques: Gene Expression, Sequencing, Expressing, Staining, In Vivo, Isolation, Western Blot, In Vitro, Fluorescence, Double Staining, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Schwann cell-secreted frizzled-related protein 1 dictates neuroinflammation and peripheral nerve degeneration after neurotrauma

doi: 10.1016/j.xcrm.2024.101791

Figure Lengend Snippet: Mice with deletion of sFRP1 in SCs profoundly reduced macrophage infiltration and improved nerve regeneration (A) Sfrp1 flox/flox mice were bred with Plpcre Ert1 mice to generate tamoxifen-inducible SC-specific sFRP1 knockout ( Sfrp1 flox/flox Plpcre Ert1 ) and littermate control ( Sfrp1 flox/flox ) mice. (B and C) Representative SCG10 immunostaining and related quantification of sciatic nerves at 14 days post transection. N = 6 mice. The dashed line indicates the transection site. Scale bar, 500 μm. (D and E) Representative F4/80 immunostaining (red) of sciatic nerves taken from the injury site, 1,000, 2,000, and 3,000 μm distal to the injury site and related quantification of infiltrated macrophages. Scale bar, 100 μm. N = 6 mice. (F and G) Western blot analysis and related quantification of TNF-α level in injured nerves at 24 h post transection. N = 3 mice. (H and I) Triple staining of CCL2 (green), F4/80 (red), and NeuN (pink) on sciatic DRG sections from Sfrp1 flox/flox and Sfrp1 flox/flox Plpcre Ert1 mice and related quantification of CCL expression level in DRGs. N = 6 mice. No significant difference of CCL2 expression is observed between DRGs of Sfrp1 flox/flox and Sfrp1 flox/flox Plpcre Ert1 mice. (J–L) Representative TUBB3 immunostaining (green) of sciatic DRG neurons isolated from Sfrp1 flox/flox and Sfrp1 flox/flox Plpcre Ert1 mice ( n = 6 mice) and related quantification. DRG neurons were cultured in vitro for 4 days or 7 days. (M and N) Representative immunostaining and related quantification of ATF3 (red) and the neuronal marker NeuN (green) in sciatic DRGs at 24 h after nerve injury. N = 6 mice. Scale bar, 50 μm. Statistical significance in (C) and (E) was analyzed by two-way ANOVA followed by Sidak’s post hoc analysis. Statistical significance was determined using two-tailed unpaired Student’s t tests; ∗∗∗∗ p < 0.0001; ∗∗∗ p < 0.001; ∗∗ p < 0.01; ∗ p < 0.05; ns, no significance. Data were presented as mean ± SD.

Article Snippet: BMDMs were lysed in RIPA lysis buffer and incubated with His-labeled sFRP1 (Ag24844, Proteintech).

Techniques: Knock-Out, Control, Immunostaining, Western Blot, Staining, Expressing, Isolation, Cell Culture, In Vitro, Marker, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Schwann cell-secreted frizzled-related protein 1 dictates neuroinflammation and peripheral nerve degeneration after neurotrauma

doi: 10.1016/j.xcrm.2024.101791

Figure Lengend Snippet: SFRP1 induces the F4/80 + CD86 + proinflammatory macrophage phenotype and inhibits oxidative metabolism (A and B) The axon length of sciatic DRG neurons demonstrates no significant difference in response to sFRP1 treatment. N = 6 biological replicates. (C) Representative TEM images reveal that the morphology and structure of mitochondria were well preserved in sFRP1-treated neurons. (D and E) Representative TEM images and related quantification of nerve transections ( N = 6 mice). The suppressing effect of sFRP1 on axon regrowth is alleviated in a macrophage-deficient condition. (F) Double staining of IL-1β (red) and TNF-α (green) on sFRP1-treated BMDMs. (G) sFRP1-induced phenotypic switch is revealed by flow cytometric quantification. FITC reflects F4/80-positive cells. PE reflects CD206-positive cells. APC reflects CD86-positive cells. (H and I) Quantification of the percentage of IL-1β and TNF-α-positive cells as reflected by Figure 4 F. Biological replicates n = 3 with two technical replicates each. (J) Double staining of Arg-1 (red) and Wnt3a (green) on sFRP1 and PBS-treated BMDMs. (K) The internalizing capacity of BMDMs was measured by incubating with 100 μg/mL pHrodo BioParticles (green). BMDMs were visualized by F4/80 (red) staining. (L and M) Quantification of the percentage of proinflammatory and pro-resolving macrophages as reflected by Figure 4 G. Biological replicates n = 3 with two technical replicates each. (N) Heatmap of differentially expressed genes between sFRP1-treated and PBS-treated macrophages (control) based on RNA sequencing. N = 3 biological replicates. (O) GO classification of differentially expressed genes related to the biological process, cellular component, and molecular function categories. (P) KEGG enrichment analysis of differentially expressed genes based on RNA sequencing. (Q) Schematic diagram of the detection of mitochondrial respiration and glycolysis of macrophages by measuring OCR and ECAR. (R) OCR of macrophages at baseline and after serial administration with oligomycin, FCCP, and rotenone plus Antimycin A. Macrophages were treated with 50, 100, 200, and 500 nM sFRP1. (S) ECAR was compared at baseline and after serial administration with glucose, oligomycin, and 2-DG. Statistical significance in (E), (H), and (I) was determined using one-way ANOVA followed by Tukey’s multiple comparisons tests; ∗∗∗∗ p < 0.0001 versus PBS group; ∗∗ p < 0.01 versus PBS group. Statistical significance in (B), (L), and (M) was determined using two-tailed unpaired Student’s t tests; ∗∗∗ p < 0.001 versus PBS group; ns, no significance. Data were presented as mean ± SD.

Article Snippet: BMDMs were lysed in RIPA lysis buffer and incubated with His-labeled sFRP1 (Ag24844, Proteintech).

Techniques: Double Staining, Staining, Control, RNA Sequencing, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Schwann cell-secreted frizzled-related protein 1 dictates neuroinflammation and peripheral nerve degeneration after neurotrauma

doi: 10.1016/j.xcrm.2024.101791

Figure Lengend Snippet: Identification of HSP90 as a binding protein to mediate the proinflammatory effect of sFRP1 on BMDMs (A) List of candidates with top 10 scores in LC-MS/MS analysis of BMDM-derived proteins with incubation of His-labeled sFRP1. (B) IP-MS analysis identifies HSP90 as an interacting protein that binds sFRP1. (C and D) IP analysis of Myc-sFRP1 (C) and HA-HSP90 (D) binding. (E) BMDMs were treated with sFRP1 plus HSP90-siRNA or control. HSP90 and sFRP1 interactions are confirmed in BMDM lysates by IP with anti-HSP90, followed by western blot analysis with anti-HSP90 and anti-sFRP1 antibody, respectively. (F) Representative IHC images of human nerves stained for HSP90 at 12 h after injury. (G) t-SNE plots of injured nerves marked by genes of HSP90 isoforms. Color key from orange to yellow indicated relative gene expression levels from high to low. (H–J) Representative IF staining and related quantification of TNF-α (red), F4/80 (red), and p65 (green) staining on BMDMs treated with sFRP1 plus HSP90-siRNA and controls. Biological replicates n = 3 with two technical replicates each. (K–M) BMDM phenotypic switch as revealed by flow cytometric quantification. FITC reflects F4/80-positive cells. PE reflects CD206-positive cells. APC reflects CD86-positive cells. N = 6 biological replicates. (N–P) TUBB3 staining on sciatic DRG neurons cocultured with macrophages for 4 days and 7 days and related quantification of axonal length. Scale bar, 100 μm. The start and the end of an axon were marked by red arrows. Biological replicates n = 3 with two technical replicates each. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons tests; ∗∗∗∗ p < 0.0001; ∗∗∗ p < 0.001; ∗∗ p < 0.01; ns, no significance. Data were presented as mean ± SD.

Article Snippet: BMDMs were lysed in RIPA lysis buffer and incubated with His-labeled sFRP1 (Ag24844, Proteintech).

Techniques: Binding Assay, Liquid Chromatography with Mass Spectroscopy, Derivative Assay, Incubation, Labeling, Protein-Protein interactions, Control, Western Blot, Staining, Gene Expression

Journal: Cell Reports Medicine

Article Title: Schwann cell-secreted frizzled-related protein 1 dictates neuroinflammation and peripheral nerve degeneration after neurotrauma

doi: 10.1016/j.xcrm.2024.101791

Figure Lengend Snippet: Depletion of HSP90 in macrophages attenuated neuroinflammation and nerve degenerative changes exerted by sFRP1 (A) Hsp90aa flox/+ mice were bred with Lyz2-cre mice to generate macrophage-specific HSP90-deficient ( Hsp90aa flox/+ Lyz2-cre ) and littermate control ( Hsp90aa flox/+ ) mice. (B and C) Representative IF images of SCG10 staining and related quantification of sciatic nerves at 2 weeks post injury. The dashed line indicates the transection site. Scale bar, 500 μm. N = 6 mice. (D and E) Representative IF images of F4/80 staining (red) of sciatic nerves and related quantification of macrophages at 2 weeks post injury. Scale bar, 100 μm. N = 6 mice. (F–I) Double staining of TNF-α (red) and IL-1β (green) on nerve longitudinal sections and related quantification. (J–L) Representative TUBB3 staining (green) and related quantification of sciatic DRG neurons isolated from Hsp90aa flox/+ and Hsp90aa flox/+ Lyz2-cre mice after 4 days and 7 days of culture. Biological replicates n = 3 with two technical replicates each. Statistical significance was determined using two-way ANOVA followed by Sidak’s post hoc analysis in (C) and (E), and using two-tailed unpaired Student’s t tests in (F), (G), (K), and (L); ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗ p < 0.05; ns, no significance. Data were presented as mean ± SD.

Article Snippet: BMDMs were lysed in RIPA lysis buffer and incubated with His-labeled sFRP1 (Ag24844, Proteintech).

Techniques: Control, Staining, Double Staining, Isolation, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Schwann cell-secreted frizzled-related protein 1 dictates neuroinflammation and peripheral nerve degeneration after neurotrauma

doi: 10.1016/j.xcrm.2024.101791

Figure Lengend Snippet: SFRP1-neutralizing antibody treatment improved axon regeneration in vivo and in vitro (A and B) Representative SCG10 immunostaining and related quantification of murine injured nerves at 2 weeks after nerve transection. The dashed line indicates the transection site. Scale bar, 500 μm. N = 6 mice. (C) Schematic diagram of DRG neuron and macrophage microfluidic coculture chamber assay. (D) Representative optical images of macrophages in the neuron-macrophage coculture chambers. (E and F) Representative TUBB3 immunofluorescent images of neurons in the neuron-macrophage co-culture chambers and related quantification of average axon length in microfluidic channels. Biological replicates n = 3 with two technical replicates each. (G) Schematic diagram of DRG neuron and macrophage direct coculture assay. (H and I) Representative IF images stained for TUBB3 (green) on sciatic DRG neurons, and quantification of average axon length per cell in the direct coculture dishes. Biological replicates n = 3 with two technical replicates each. Statistical significance was determined using two-way ANOVA followed by Sidak’s post hoc analysis in (B) and (I) and using two-tailed unpaired Student’s t tests in (F); ∗∗∗ p < 0.001; ∗∗ p < 0.01; ∗ p < 0.05. Data were presented as mean ± SD.

Article Snippet: BMDMs were lysed in RIPA lysis buffer and incubated with His-labeled sFRP1 (Ag24844, Proteintech).

Techniques: In Vivo, In Vitro, Immunostaining, Boyden Chamber Assay, Co-Culture Assay, Co-culture Assay, Staining, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Schwann cell-secreted frizzled-related protein 1 dictates neuroinflammation and peripheral nerve degeneration after neurotrauma

doi: 10.1016/j.xcrm.2024.101791

Figure Lengend Snippet:

Article Snippet: BMDMs were lysed in RIPA lysis buffer and incubated with His-labeled sFRP1 (Ag24844, Proteintech).

Techniques: Cytometry, Recombinant, Sequencing, Plasmid Preparation, Software

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Wnt1 neuroprotection translates into improved neurological function during oxidant stress and cerebral ischemia through AKT1 and mitochondrial apoptotic pathways

doi: 10.4161/oxim.3.2.9

Figure Lengend Snippet: OGD leads to progressive injury in neurons and reduces endogenous Wnt1 expression over time. In A, primary hippocampal neurons were exposed to progressive durations of OGD at 1, 2, 3 and 4 hours and neuronal survival was determined 24 hours later by trypan blue dye exclusion assay. Neuronal survival was decreased to 73 ± 3% (1 hour), 51 ± 4% (2 hours), 32 ± 3% (3 hours), and 20 ± 3% (4 hours) following OGD exposure when compared with untreated control cultures (86 ± 3%, *p < 0.01 vs. Control). Each data point represents the mean and SEM from 6 experiments. In B, neuronal protein extracts (50 µg/lane) were immunoblotted with anti-Wnt1 (Wnt1) at 1, 6 and 24 hours following OGD exposure. Wnt1 expression is initially elevated at 1 hour and 6 hours, but then progressively and significantly is reduced at 24 hours following OGD exposure (*p < 0.01 vs. control)

Article Snippet: For treatments applied prior to OGD, human recombinant Wnt1 protein (R&D Systems, Minneapolis, MN) or mouse monoclonal anti body against Wnt1 (Wnt1Ab) (R&D Systems, Minneapolis, MN), the phosphatidylinositol 3-kinase (PI-3 K) inhibitor wortmannin (Calbiochem, La Jolla, CA), the recombinant Wnt antagonist dickkopf related protein 1 (DKK-1, 0.5 μg/ml, R&D Systems, Minneapolis, MN), or D-3-deoxy-2-O-methyl- myo inositol 1-(R)-2-methoxy-3-(octadecyloxy) propyl hydrogen phosphate (SH-5) (Alexis, San Diego, CA) were continuous.

Techniques: Expressing, Exclusion Assay, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Wnt1 neuroprotection translates into improved neurological function during oxidant stress and cerebral ischemia through AKT1 and mitochondrial apoptotic pathways

doi: 10.4161/oxim.3.2.9

Figure Lengend Snippet: Transient transfection of Wnt1 increases neuronal survival and prevents genomic DNA degradation and membrane ps externalization. In A, overexpression of Wnt1 was performed under the control of a CMV promoter with Wnt1 cDNA and cell survival was determined 24 hours after OGD exposure through the trypan blue dye exclusion method. Representative images illustrate decreased trypan blue staining during Wnt1 transient transfection. Significant cell injury and trypan blue staining occurs during OGD alone in wildtype cells and during vector transfection. Quantification of data demonstrates that OGD significantly decreased percent cell survival when compared to the control cells. In contrast, Wnt1 significantly increased cell survival (*p < 0.01 vs. OGD). Each data point represents the mean and SEM from six experiments. Control = untreated neurons. In B, overexpression of Wnt1 was performed under the control of a CMV promoter with Wnt1 cDNA and genomic DNA degradation was determined 24 hours after OGD exposure through TUNEL. Representative images illustrate decreased TUNEL staining during Wnt1 transient transfection. Significant DNA fragmentation occurs during OGD alone in wildtype cells and during vector transfection. Quantification of data demonstrates that OGD results in marked DNA fragmentation when compared to the control cells. In contrast, Wnt1 significantly prevents DNA fragmentation (*p < 0.01 vs. OGD). Each data point represents the mean and SEM from six experiments. Control = untreated neurons. In C, overexpression of Wnt1 was performed under the control of a CMV promoter with Wnt1 cDNA and membrane PS externalization was determined 24 hours after OGD exposure through annexin V phycoerythrin (green fluorescence). Representative images illustrate decreased PS staining during Wnt1 transient transfection. Significant membrane PS externalization occurs during OGD alone in wildtype cells and during vector transfection. Quantification of data demonstrates that OGD results in marked PS exposure when compared to the control cells. In contrast, Wnt1 significantly prevents PS externalization (*p < 0.01 vs. OGD). Each data point represents the mean and SEM from six experiments. Control, untreated neurons.

Article Snippet: For treatments applied prior to OGD, human recombinant Wnt1 protein (R&D Systems, Minneapolis, MN) or mouse monoclonal anti body against Wnt1 (Wnt1Ab) (R&D Systems, Minneapolis, MN), the phosphatidylinositol 3-kinase (PI-3 K) inhibitor wortmannin (Calbiochem, La Jolla, CA), the recombinant Wnt antagonist dickkopf related protein 1 (DKK-1, 0.5 μg/ml, R&D Systems, Minneapolis, MN), or D-3-deoxy-2-O-methyl- myo inositol 1-(R)-2-methoxy-3-(octadecyloxy) propyl hydrogen phosphate (SH-5) (Alexis, San Diego, CA) were continuous.

Techniques: Transfection, Membrane, Over Expression, Control, Staining, Plasmid Preparation, TUNEL Assay, Fluorescence

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Wnt1 neuroprotection translates into improved neurological function during oxidant stress and cerebral ischemia through AKT1 and mitochondrial apoptotic pathways

doi: 10.4161/oxim.3.2.9

Figure Lengend Snippet: Wnt1 signaling is necessary for neuronal protection against OGD. In A, increasing concentrations of Wnt1 protein result in significantly increased neuronal survival assessed by trypan blue exclusion 24 hours after OGD (*p < 0.01 vs. OGD). In B, increasing concentrations of an antibody to Wnt1 (Wnt1Ab) during OGD do not alter neuronal survival assessed by trypan blue exclusion 24 hours after OGD when compared to neurons exposed to OGD alone. In C, increasing concentrations of an antibody to Wnt1 (Wnt1Ab) applied with Wnt1 (100 ng/ml) resulted in progressive loss of Wnt1 protection and increased neuronal cell injury assessed by trypan blue staining 24 hours after OGD (*p < 0.01 vs. OGD). In D, inhibition of Wnt1 signaling with DKK-1 (0.5 µg/ml), an antagonist of the Wnt/β-catenin pathway, administered with Wnt1 (100 ng/ml) significantly reduces protection by Wnt1 and neuronal cell survival assessed by trypan blue staining 24 hours after OGD (*p < 0.01 vs. OGD; †p < 0.01 vs. Wnt1/OGD). In all cases control = untreated neurons. Each data point represents the mean and SEM from six experiments.

Article Snippet: For treatments applied prior to OGD, human recombinant Wnt1 protein (R&D Systems, Minneapolis, MN) or mouse monoclonal anti body against Wnt1 (Wnt1Ab) (R&D Systems, Minneapolis, MN), the phosphatidylinositol 3-kinase (PI-3 K) inhibitor wortmannin (Calbiochem, La Jolla, CA), the recombinant Wnt antagonist dickkopf related protein 1 (DKK-1, 0.5 μg/ml, R&D Systems, Minneapolis, MN), or D-3-deoxy-2-O-methyl- myo inositol 1-(R)-2-methoxy-3-(octadecyloxy) propyl hydrogen phosphate (SH-5) (Alexis, San Diego, CA) were continuous.

Techniques: Staining, Inhibition, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Wnt1 neuroprotection translates into improved neurological function during oxidant stress and cerebral ischemia through AKT1 and mitochondrial apoptotic pathways

doi: 10.4161/oxim.3.2.9

Figure Lengend Snippet: Wnt1 relies upon the PI 3-K pathway and Akt1 to provide neuronal protection. In A, primary neuronal protein extracts (50 µ/lane) were immunoblotted with anti-phosphorylated-Akt1 (p-Akt1, Ser 473 ) or anti-total Akt1 at 6 hours following OGD. Application of Wnt1 (100 ng/ml) in untreated wildtype neurons or in the presence of OGD significantly elevated p-Akt1 expression to a greater extent than OGD alone. This increased expression of p-Akt1 by Wnt1 was blocked by the PI 3-K inhibitor wortmannin (0.5 µM) and by the specific Akt1 inhibitor SH-5 (20 µM). Total Akt1 is not altered (*p < 0.01, vs. OGD; †p < 0.01 vs. Wnt1/OGD). Each data point represents the mean and SEM from six experiments. In B, primary neurons treated with Wnt1 (100 ng/ml) increased neuronal survival assessed by trypan blue staining 24 hours after OGD. Yet, application of wortmannin (0.5 µM) or SH-5 (20 µM) at concentrations that block activation of Akt1 activation significantly reduced protection by Wnt1 24 hours after OGD (*p < 0.01, vs. OGD; †p < 0.01 vs. Wnt1/OGD). Each data point represents the mean and SEM from six experiments.

Article Snippet: For treatments applied prior to OGD, human recombinant Wnt1 protein (R&D Systems, Minneapolis, MN) or mouse monoclonal anti body against Wnt1 (Wnt1Ab) (R&D Systems, Minneapolis, MN), the phosphatidylinositol 3-kinase (PI-3 K) inhibitor wortmannin (Calbiochem, La Jolla, CA), the recombinant Wnt antagonist dickkopf related protein 1 (DKK-1, 0.5 μg/ml, R&D Systems, Minneapolis, MN), or D-3-deoxy-2-O-methyl- myo inositol 1-(R)-2-methoxy-3-(octadecyloxy) propyl hydrogen phosphate (SH-5) (Alexis, San Diego, CA) were continuous.

Techniques: Expressing, Staining, Blocking Assay, Activation Assay

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Wnt1 neuroprotection translates into improved neurological function during oxidant stress and cerebral ischemia through AKT1 and mitochondrial apoptotic pathways

doi: 10.4161/oxim.3.2.9

Figure Lengend Snippet: Wnt1 uses Akt1 to block apoptotic membrane PS exposure and genomic DNA degradation during OGD. In A, representative images illustrate that recombinant human Wnt1 protein (100 ng/ml) significantly blocks neuronal genomic DNA degradation assessed by TUNEL and membrane PS externalization assessed by annexin V phycoerythrin (green fluorescence) 24 hours following OGD. In contrast, inhibition of Wnt1 (100 ng/ml) signaling with Wnt1Ab (1 µg/ml) or with application of DKK-1 (0.5 µg/ml) or inhibition of Akt1 with SH-5 (20 µM) during Wnt1 application leads to the loss of Wnt1 protection. In B, quantification of data illustrates that DNA fragmentation and membrane PS externalization were significantly increased 24 hours following OGD when compared to untreated neuronal control cultures during Wnt1 (100 ng/ml) application with Wnt1Ab (1 µg/ml), DKK-1 (0.5 µg/ml), or SH-5 (20 µM). Each data point represents the mean and SEM from six experiments.

Article Snippet: For treatments applied prior to OGD, human recombinant Wnt1 protein (R&D Systems, Minneapolis, MN) or mouse monoclonal anti body against Wnt1 (Wnt1Ab) (R&D Systems, Minneapolis, MN), the phosphatidylinositol 3-kinase (PI-3 K) inhibitor wortmannin (Calbiochem, La Jolla, CA), the recombinant Wnt antagonist dickkopf related protein 1 (DKK-1, 0.5 μg/ml, R&D Systems, Minneapolis, MN), or D-3-deoxy-2-O-methyl- myo inositol 1-(R)-2-methoxy-3-(octadecyloxy) propyl hydrogen phosphate (SH-5) (Alexis, San Diego, CA) were continuous.

Techniques: Blocking Assay, Membrane, Recombinant, TUNEL Assay, Fluorescence, Inhibition, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Wnt1 neuroprotection translates into improved neurological function during oxidant stress and cerebral ischemia through AKT1 and mitochondrial apoptotic pathways

doi: 10.4161/oxim.3.2.9

Figure Lengend Snippet: Wnt1 inhibits mitochondrial depolarization and prevents the release of cytochrome c during OGD. In A, OGD leads to a significant decrease in the red/green fluorescence intensity ratio of mitochondria using the cationic membrane potential indicator JC-1 within 3 hours when compared with untreated control neurons, demonstrating that OGD leads to mitochondrial membrane depolarization. Application of Wnt1 (100 ng/ml) during OGD significantly increased the red/green fluorescence intensity of mitochondria in neurons, illustrating that mitochondrial membrane potential was restored by Wnt1. In contrast, inhibition of Wnt1 (100 ng/ml) signaling with Wnt1Ab (1 µg/ml) or with application of DKK-1 (0.5 µg/ml) or inhibition of Akt1 with SH-5 (20 µM) during Wnt1 application and OGD resulted in mitochondrial depolarization similar to OGD exposure alone. In B, the relative ratio of red/green fluorescent intensity of mitochondrial staining in untreated control neurons, in neurons exposed to OGD, during Wnt1 (100 ng/ml)/OGD application alone or during Wnt1 (100 ng/ml)/OGD with Wnt1Ab (1 µg/ml), DKK-1 (0.5 µg/ml), or SH-5 (20 µM) was measured in six independent experiments with analysis performed using the public domain NIH Image program ( http://rsb.info.nih.gov/nih-image ) (Wnt1/OGD vs. OGD, *p < 0.01; Wnt1/OGD with Wnt1Ab, DKK-1, or SH-5 vs. Wnt1/OGD, †p < 0.01). In C and D, equal amounts of mitochondrial (mito) or cytosol (cyto) protein extracts (50 µg/lane) were immunoblotted demonstrating that Wnt1 (100 ng/ml) significantly prevented cytochrome c release from mitochondria within 3 hours after OGD but Wnt1Ab (1 µg/ml), DKK-1 (0.5 µg/ml), or SH-5 (20 µM) during Wnt1/OGD application prevented Wnt1 from maintaining cytochrome c in the mitochondria (Wnt1/OGD vs. OGD, *p < 0.01; Wnt1/OGD with Wnt1Ab, DKK-1, or SH-5 vs. Wnt1/OGD, †p < 0.01).

Article Snippet: For treatments applied prior to OGD, human recombinant Wnt1 protein (R&D Systems, Minneapolis, MN) or mouse monoclonal anti body against Wnt1 (Wnt1Ab) (R&D Systems, Minneapolis, MN), the phosphatidylinositol 3-kinase (PI-3 K) inhibitor wortmannin (Calbiochem, La Jolla, CA), the recombinant Wnt antagonist dickkopf related protein 1 (DKK-1, 0.5 μg/ml, R&D Systems, Minneapolis, MN), or D-3-deoxy-2-O-methyl- myo inositol 1-(R)-2-methoxy-3-(octadecyloxy) propyl hydrogen phosphate (SH-5) (Alexis, San Diego, CA) were continuous.

Techniques: Fluorescence, Membrane, Control, Inhibition, Staining

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Wnt1 neuroprotection translates into improved neurological function during oxidant stress and cerebral ischemia through AKT1 and mitochondrial apoptotic pathways

doi: 10.4161/oxim.3.2.9

Figure Lengend Snippet: Transient cerebral ischemia blocks endogenous Wnt1 cortical expression but exogenous Wnt1 is protective against cerebral ischemia. In A and B, focal cerebral ischemia was induced by insertion of a monofilament thread (4-0) into the internal carotid artery and blockade of the origin of MCA. Reperfusion was performed following 90 minutes ischemia by withdrawal of the thread. For A, cortical protein extracts (50 µg/lane) were immunoblotted with anti-Wnt1 (Wnt1) at 1, 6 and 24 hours following OGD exposure. Similar to the effects upon Wnt1 expression with OGD in neuronal cultures, Wnt1 expression is initially elevated at 1 hour and 6 hours, but then progressively and significantly is reduced at 24 hours following OGD exposure (*p < 0.01 vs. control). Wnt1 expression on the contralateral non-infarction hemisphere was not altered from control, illustrating that the generation of cerebral ischemia directly led to changes in endogenous Wnt1 expression. In B, Wnt1 protein (24 µg/kg) was injected into the internal carotid artery through the external carotid artery at 30 minutes prior to the onset of MCAO and at the onset of reperfusion. Animals were euthanized 24 hours following ischemia and the infarct size was determined by 2,3,5-triphenytetrazolium (TTC) staining. Representative images show that the infarct size (white in color) was significantly reduced by treatment with Wnt1 protein. In C, quantitative results demonstrate that infarct size was significantly decreased by Wnt1 (24 µg/kg) treatment following reperfusion after cerebral ischemia. The total infarct size was expressed as a percentage of the contralateral hemisphere (*p < 0.05 vs. vehicle). In all cases, each data point represents the mean and SEM from six experiments.

Article Snippet: For treatments applied prior to OGD, human recombinant Wnt1 protein (R&D Systems, Minneapolis, MN) or mouse monoclonal anti body against Wnt1 (Wnt1Ab) (R&D Systems, Minneapolis, MN), the phosphatidylinositol 3-kinase (PI-3 K) inhibitor wortmannin (Calbiochem, La Jolla, CA), the recombinant Wnt antagonist dickkopf related protein 1 (DKK-1, 0.5 μg/ml, R&D Systems, Minneapolis, MN), or D-3-deoxy-2-O-methyl- myo inositol 1-(R)-2-methoxy-3-(octadecyloxy) propyl hydrogen phosphate (SH-5) (Alexis, San Diego, CA) were continuous.

Techniques: Expressing, Control, Injection, Staining

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Wnt1 neuroprotection translates into improved neurological function during oxidant stress and cerebral ischemia through AKT1 and mitochondrial apoptotic pathways

doi: 10.4161/oxim.3.2.9

Figure Lengend Snippet: Wnt1 signaling is required for reduction in cerebral infarction following transient cerebral MCAO and maintains neurological recovery. In A, B and C, focal cerebral ischemia was induced by insertion of a monofilament thread (4-0) into the internal carotid artery and blockade of the origin of MCA. Reperfusion was performed following 90 minutes ischemia by withdrawal of the thread. In A, Wnt1 protein (24 µg/kg), Wnt1Ab (60 µg/kg), or DKK-1 (30 µg/kg) was injected into the internal carotid artery through the external carotid artery at 30 min prior to the onset of MCAO and at the onset of reperfusion. Animals were euthanized 24 hours following ischemia and infarct size was determined by 2,3,5-triphenytetrazolium (TTC) staining. Representative images illustrate that infarct size (white in color) was significantly reduced by treatment with Wnt1 protein. In contrast, infarct size was markedly increased by treatment with Wnt1Ab or DKK-1 during MCAO. In B, quantitative results demonstrate that infarct size was significantly decreased by Wnt1 (24 µg/kg) treatment following reperfusion after MCAO. However, infarct size was substantially increased with Wnt1Ab (60 µg/kg) or DKK-1 (30 µg/kg) administration during MCAO. The total infarct size was expressed as a percentage of the contralateral hemisphere (*p < 0.05 vs. vehicle). In C, the neurological deficit score was assessed in animals 24 hours following MCAO and reperfusion of a 90 minute period. Wnt1 (24 µg/kg) significantly lowered the neurological deficit score when compared to vehicle only treated animals. In contrast, Wnt1Ab (60 µg/kg) or DKK-1 (30 µg/kg) significantly increased the neurological deficit score (*p < 0.05 vs. vehicle). In all cases, each data point represents the mean and SEM from six experiments.

Article Snippet: For treatments applied prior to OGD, human recombinant Wnt1 protein (R&D Systems, Minneapolis, MN) or mouse monoclonal anti body against Wnt1 (Wnt1Ab) (R&D Systems, Minneapolis, MN), the phosphatidylinositol 3-kinase (PI-3 K) inhibitor wortmannin (Calbiochem, La Jolla, CA), the recombinant Wnt antagonist dickkopf related protein 1 (DKK-1, 0.5 μg/ml, R&D Systems, Minneapolis, MN), or D-3-deoxy-2-O-methyl- myo inositol 1-(R)-2-methoxy-3-(octadecyloxy) propyl hydrogen phosphate (SH-5) (Alexis, San Diego, CA) were continuous.

Techniques: Injection, Staining

Journal: bioRxiv

Article Title: Fgf9-Nolz-1-Wnt2 Signaling Axis Regulates Morphogenesis of the Lung

doi: 10.1101/2022.08.10.503529

Figure Lengend Snippet: (A) Morphogenic molecules are screened by qRT-PCR in E12.5 Nolz-1 mutant lungs. Wnt2 and Lef1 mRNA are decreased in Nolz-1 mutant lungs. Fgf10 is decreased and Pdgfrα is increased in the Nolz-1 mutant lungs. Gli3 and Bmp4 are decreased and Tgf-β2 is increased in Nolz-1 mutant lungs. Student’s t- test, * P < 0.05, ** P < 0.01, n = 3. (B) In situ hybridization shows that Wnt2 mRNA is mainly expressed in the distal parts of the mesenchyme of E11.5 and E13.5 wild type lungs. Wnt2 mRNA is markedly decreased in mutant mesenchyme. The insets show high magnification of the regions indicated by asterisks. Scale bars, 50 μm. (C) The qRT-PCR assay shows that over-expression of Nolz-1 by electroporation of pcBIG-myc-Nolz-1-ires-EGFP plasmid up-regulates Wnt2 , cyclinD1 and c-myc mRNAs in primary mesenchymal cell culture derived from E14.5 wild type mouse lungs compared to mock transfection of pcBIG-ires-EGFP control plasmids. Student’s t- test, * P < 0.05, *** P < 0.001, n = 4. (D) The schematic drawing illustrates the locations of the putative Nolz-1 binding sites of “AGGAT” at -788 (N1 motif) and -2194 (N2 motif) in 5 ‘flânking regions of mouse Wnt2 gene (+1: ATG translation start site). (E) The chromatin immunoprecipitation (ChIP) assay shows that a 187 bp PCR band is amplified from the N2 locus with the immunoprecipitated products using the anti-myc antibody, but not the control rabbit IgG, in E14.5 lung mesenchymal cell culture electroporated with pcBIG-myc-Nolz-1-ires-EGFP plasmids. No specific PCR band is detected from the N1 locus. Student’s t- test, * P < 0.05, n.s. not significant, n = 3. (F) The reporter gene assay showed that the luciferase activity is increased in the pGL3-N2-Luc group compared to the pGL3-Luc control group in E14.5 lung mesenchymal cells transfected with pcBIG-myc-Nolz-1-ires-EGFP plasmids. Student’s t- test, ** P < 0.01, n = 6.

Article Snippet: The lung explants were cultured with recombinant human Wnt2 protein (150 ng/ml, Novus Biologicals).

Techniques: Quantitative RT-PCR, Mutagenesis, In Situ Hybridization, Over Expression, Electroporation, Plasmid Preparation, Cell Culture, Derivative Assay, Transfection, Control, Binding Assay, Chromatin Immunoprecipitation, Amplification, Immunoprecipitation, Reporter Gene Assay, Luciferase, Activity Assay

Journal: bioRxiv

Article Title: Fgf9-Nolz-1-Wnt2 Signaling Axis Regulates Morphogenesis of the Lung

doi: 10.1101/2022.08.10.503529

Figure Lengend Snippet: (A) Treatment with rFgf10 (200 n /ml) of wild type explant lung culture for 48 hr. qRT-PCR shows that Nolz-1 and Wnt2 are not changed in the rFgf10 treated group compared to the vehicle control. Student’s t -test, P > 0.05, n = 4. Scale bar, 500 μm. (B) Treatment with rFgf9 (200 ng/ml) results in enlarged epithelia in wild-type explant lungs cultured for 48 hr. The qRT-PCR shows that Nolz-1 , Wnt2 and Lef1 mRNAs are increased in rFgf9 treated group in wild type lungs. Student’s t -test, * P < 0.05, ** P < 0.01, n = 4. Western blotting showed that rFgf9 treatment increases Nolz-1 protein by 99% in wild type lung culture compared to the vehicle-treated group. Student’s t -test, * P < 0.05, n = 3. (C) Working hypothesis. Nolz-1 controls the proliferation of mesenchymal cells and the growth of epithelial branches through the regulation of Wnt2 signaling in the early stages of the development of the lungs. In the late stages of development, Nolz-1 acts non-cell autonomously to regulate the development of epithelial cells through Wnt2 signaling. Fgf9 acts upstream to regulate Nolz-1 expression in developing lungs.

Article Snippet: The lung explants were cultured with recombinant human Wnt2 protein (150 ng/ml, Novus Biologicals).

Techniques: Quantitative RT-PCR, Control, Cell Culture, Western Blot, Expressing