Journal: bioRxiv

Article Title: Compound screening in primary human airway basal cells identifies Wnt pathway activators as potential pro-regenerative therapies

doi: 10.1101/2024.08.13.606573

Figure Lengend Snippet: A) Quantification of Wnt activity using the TOPFlash assay. HBEC3-KT cells were transfected with TOPFlash plasmid, re-plated in a 384-well plate and incubated with 10 μM of the indicated compounds for 24 hours. An ANOVA was performed and a Wilcoxon t-test with Holm correction for multiple testing to compare control and compound treated wells. ** = p<0.01. B) Quantification of Wnt activity using the TOPFlash assay in HBEC3-KT cells treated with kenpaullone, 1-azakenpaullone, BIO, or BIO and iCRT14 (a Wnt pathway inhibitor) for 24 hours. Negative control FOPFlash assays performed in parallel were negative as expected (not shown). An ANOVA was performed and a Wilcoxon t-test with Holm correction for multiple testing to compare control and compound treated wells. * = p<0.05. C) qPCR analysis of the Wnt target genes LEF1 and AXIN2 . Primary human airway basal cells (n = 4 donors) were treated with compounds for 24 hours. Relative expression was determined by normalising to the reference genes RPS13 and GAPDH . An ANOVA test was performed per target gene and significant Tukey’s HSD values are shown. D) Quantification of primary human airway basal cell proliferation over six days using the CellTiter-Glo assay. Data are normalised to control wells containing the highest concentration of DMSO used in the experimental conditions. Points are the mean value of quadruplicate wells per donor (n = 5 donors). E) Effect of the Wnt pathway inhibitor iCRT14 on the proliferation of airway basal cells in the presence of kenpaullone, 1-azakenpaullone or BIO. Cells were pre-treated with 10 μM iCRT14 or DMSO control for 24 hours before the addition of the Wnt activating compounds. Relative growth was assessed after two days using the CellTiter-Glo assay (n = 4 donors). Wilcoxon tests were performed with Holm correction for multiple testing per compound, ****p<0.0001. F) Quantification of mean organoid size per well with 12 replicate wells per condition were normalised to mean control well organoid size for each donor (n = 5 donors [ID2a = circles, ID5 = triangles, ID6 = squares, ID7 = cross, ID8 = checked box]. An ANOVA was performed per compound and significant Tukey’s HSD values are shown. ** = p<0.01, **** = p<0.0001). Control well data are repeated per compound facet and kenpaullone data is repeated from . G) Immunofluorescence staining showing KRT5 (basal cells, white), MUC5AC (mucosecretory cells, magenta), and ACT (ciliated cells, yellow) in organoids following culture in medium containing the indicated compounds (n=5, donor ID= 8 shown). Scale bars = 50 μm.

Article Snippet: The following day cells were co-transfected with the M50 Super 8x TOPFlash (a gift from Randall Moon, Addgene plasmid #12456; http://n2t.net/addgene:12456 ; RRID:Addgene_12456), the M51 Super 8x FOPFlash – an inactive TOPFlash mutant (a gift from Randall Moon, Addgene plasmid #12457) and the pRL SV40 construct (E2231, Promega) using jetOPTIMUS (101000051, Polyplus) according to the manufacturer’s instructions.

Techniques: Activity Assay, TOPFlash assay, Transfection, Plasmid Preparation, Incubation, Control, Negative Control, Expressing, Glo Assay, Concentration Assay, Immunofluorescence, Staining

Journal: Molecular and Cellular Neurosciences

Article Title: Neurotrophin and Wnt signaling cooperatively regulate dendritic spine formation ☆

doi: 10.1016/j.mcn.2013.04.006

Figure Lengend Snippet: Wnt inhibition results in altered dendritic spine development. (A) Representative dendritic segments of cortical neurons expressing empty vector (EV), Wif1, Sfrp1, Fzd8CRD or Dvl1ΔPDZ. (B) Quantification of dendritic protrusion density. (C) Percent of all dendritic protrusions classified as either spines or filopodia. Quantification of average protrusion length (D) and average spine head width (E) for each treatment. *** p < 0.001, ** p < 0.01, * p < 0.05. n = number of neurons: EV n = 31, Wif1 n = 34 Sfrp1 n = 23, Fzd8CRD n = 25, Dvl1ΔPDZ n = 25. Scale bar: 5 μm.

Article Snippet: pCMV-BDNF was constructed by inserting the open reading frame for murine BDNF into the pEGFP-N1 backbone (Clontech, Mountain View, CA). pBR22-Wif1 expresses murine Wif1 and was a generous gift from Dr. Ken Iwatsuki (Ajinomoto Co., Tokyo, Japan). pCS2 +-Sfrp1 (Addgene plasmid 16693, Cambridge, MA) expresses murine Sfrp1 (Randall Moon Lab). pRK5-mFzd8CRD-IgG (Addgene plasmid 16689) expresses a fusion protein consisting of the extracellular domain of the murine Frizzled-8 protein fused with the human immunoglobulin heavy chain ( ). pCS2 +-mDvl1ΔPDZ-HA expresses a C-terminally HA tagged murine Dishevelled-1 with a deletion of amino acids 276–336 ( ). pTRE-tight-BDNF and pTRE-tight-Wnt2 were constructed by inserting the ORFs for murine BDNF and murine Wnt2, respectively, into the pTRE-tight backbone (Clontech). pTRE-tight-EGFP was constructed by inserting the EGFP ORF from pEGFP-N1 (Clontech) into the pTRE-tight backbone. pCMV-rtTA was constructed by inserting the ORF for the reverse-tet-Transactivator protein from pTRIPZ (Thermo Scientific) into the pEGFP-N1 backbone from which the EGFP coding region was deleted.

Techniques: Inhibition, Expressing, Plasmid Preparation

Journal: Molecular and Cellular Neurosciences

Article Title: Neurotrophin and Wnt signaling cooperatively regulate dendritic spine formation ☆

doi: 10.1016/j.mcn.2013.04.006

Figure Lengend Snippet: Expression of Wnt inhibitors or Wnt2 shifts the distribution of dendritic protrusion lengths. (A) Relative frequency distributions of dendritic protrusion length comparing Wif1, Sfrp1, Fzd8CRD and Dvl1ΔPDZ expressing neurons to EV control. (B) Relative frequency distribution of protrusion length comparing Wnt2 expressing neurons to EV control. n = number of neurons: EV(A) n = 31, Wif1 n = 34, Sfrp1 n = 23, Fzd8CRD n = 25, Dvl1ΔPDZ n = 25; EV(B) n = 29, Wnt2 n = 25. *** p < 0.001, ** p < 0.01, * p < 0.05.

Article Snippet: pCMV-BDNF was constructed by inserting the open reading frame for murine BDNF into the pEGFP-N1 backbone (Clontech, Mountain View, CA). pBR22-Wif1 expresses murine Wif1 and was a generous gift from Dr. Ken Iwatsuki (Ajinomoto Co., Tokyo, Japan). pCS2 +-Sfrp1 (Addgene plasmid 16693, Cambridge, MA) expresses murine Sfrp1 (Randall Moon Lab). pRK5-mFzd8CRD-IgG (Addgene plasmid 16689) expresses a fusion protein consisting of the extracellular domain of the murine Frizzled-8 protein fused with the human immunoglobulin heavy chain ( ). pCS2 +-mDvl1ΔPDZ-HA expresses a C-terminally HA tagged murine Dishevelled-1 with a deletion of amino acids 276–336 ( ). pTRE-tight-BDNF and pTRE-tight-Wnt2 were constructed by inserting the ORFs for murine BDNF and murine Wnt2, respectively, into the pTRE-tight backbone (Clontech). pTRE-tight-EGFP was constructed by inserting the EGFP ORF from pEGFP-N1 (Clontech) into the pTRE-tight backbone. pCMV-rtTA was constructed by inserting the ORF for the reverse-tet-Transactivator protein from pTRIPZ (Thermo Scientific) into the pEGFP-N1 backbone from which the EGFP coding region was deleted.

Techniques: Expressing, Control

Journal: Molecular and Cellular Neurosciences

Article Title: Neurotrophin and Wnt signaling cooperatively regulate dendritic spine formation ☆

doi: 10.1016/j.mcn.2013.04.006

Figure Lengend Snippet: Wnt inhibition results in decreased dendrite elaboration. (A) Representative cortical neurons expressing EV, Wif1, Sfrp1, Fzd8CRD or Dvl1ΔPDZ. (B) Quantification of the total dendrite length per neuron for each treatment. (C) Quantification of the number of dendritic endpoints per neuron for each treatment. (D–G) Sholl analysis of dendritic complexity comparing neurons treated with each Wnt inhibitor to control neurons. *** p < 0.001, ** p < 0.01, * p < 0.05. n = number of neurons: EV n = 56, Wif1 n = 49, Sfrp1 n = 39, Fzd8CRD n = 39, Dvl1ΔPDZ n = 39. Scale bar: 50 μm.

Article Snippet: pCMV-BDNF was constructed by inserting the open reading frame for murine BDNF into the pEGFP-N1 backbone (Clontech, Mountain View, CA). pBR22-Wif1 expresses murine Wif1 and was a generous gift from Dr. Ken Iwatsuki (Ajinomoto Co., Tokyo, Japan). pCS2 +-Sfrp1 (Addgene plasmid 16693, Cambridge, MA) expresses murine Sfrp1 (Randall Moon Lab). pRK5-mFzd8CRD-IgG (Addgene plasmid 16689) expresses a fusion protein consisting of the extracellular domain of the murine Frizzled-8 protein fused with the human immunoglobulin heavy chain ( ). pCS2 +-mDvl1ΔPDZ-HA expresses a C-terminally HA tagged murine Dishevelled-1 with a deletion of amino acids 276–336 ( ). pTRE-tight-BDNF and pTRE-tight-Wnt2 were constructed by inserting the ORFs for murine BDNF and murine Wnt2, respectively, into the pTRE-tight backbone (Clontech). pTRE-tight-EGFP was constructed by inserting the EGFP ORF from pEGFP-N1 (Clontech) into the pTRE-tight backbone. pCMV-rtTA was constructed by inserting the ORF for the reverse-tet-Transactivator protein from pTRIPZ (Thermo Scientific) into the pEGFP-N1 backbone from which the EGFP coding region was deleted.

Techniques: Inhibition, Expressing, Control

Journal: Molecular and Cellular Neurosciences

Article Title: Neurotrophin and Wnt signaling cooperatively regulate dendritic spine formation ☆

doi: 10.1016/j.mcn.2013.04.006

Figure Lengend Snippet: Wnt inhibition does not affect primary dendrite number on cortical neurons. (A) Representative cell bodies of cortical neurons expressing EV, Wif1, Sfrp1, Fzd8CRD and Dvl1ΔPDZ either alone, or in combination with BDNF. (B) Quantification of the number of primary dendrites per neuron for each treatment. *** p < 0.001. n = number of neurons: EV n = 31, BDNF n = 31, Wif1 n = 34, BDNF + Wif1 n = 29, Sfrp1 n = 22, BDNF + Sfrp1 n = 21, Fzd8CRD n = 25, BDNF + Fzd8CRD n = 25, Dvl1ΔPDZ n = 25, BDNF + Dvl1ΔPDZ n = 30. Scale bar: 5 μm.

Article Snippet: pCMV-BDNF was constructed by inserting the open reading frame for murine BDNF into the pEGFP-N1 backbone (Clontech, Mountain View, CA). pBR22-Wif1 expresses murine Wif1 and was a generous gift from Dr. Ken Iwatsuki (Ajinomoto Co., Tokyo, Japan). pCS2 +-Sfrp1 (Addgene plasmid 16693, Cambridge, MA) expresses murine Sfrp1 (Randall Moon Lab). pRK5-mFzd8CRD-IgG (Addgene plasmid 16689) expresses a fusion protein consisting of the extracellular domain of the murine Frizzled-8 protein fused with the human immunoglobulin heavy chain ( ). pCS2 +-mDvl1ΔPDZ-HA expresses a C-terminally HA tagged murine Dishevelled-1 with a deletion of amino acids 276–336 ( ). pTRE-tight-BDNF and pTRE-tight-Wnt2 were constructed by inserting the ORFs for murine BDNF and murine Wnt2, respectively, into the pTRE-tight backbone (Clontech). pTRE-tight-EGFP was constructed by inserting the EGFP ORF from pEGFP-N1 (Clontech) into the pTRE-tight backbone. pCMV-rtTA was constructed by inserting the ORF for the reverse-tet-Transactivator protein from pTRIPZ (Thermo Scientific) into the pEGFP-N1 backbone from which the EGFP coding region was deleted.

Techniques: Inhibition, Expressing

Journal: Molecular and Cellular Neurosciences

Article Title: Neurotrophin and Wnt signaling cooperatively regulate dendritic spine formation ☆

doi: 10.1016/j.mcn.2013.04.006

Figure Lengend Snippet: Wnt inhibitors interfere with BDNF-induced dendritic spine formation. (A) Representative dendritic segments of cortical neurons expressing EV, BDNF with EV, BDNF with Wif1, BDNF with Sfrp1, BDNF with Fzd8CRD and BDNF with Dvl1ΔPDZ. (B) Quantification of dendritic protrusion density. (C) Fraction of all spines classified as either spines or filopodia. Quantification of average protrusion length (D) and average spine head width (E) for each treatment. *** p < 0.001, ** p < 0.01, * p < 0.5. (* = relative to EV, # = relative to EV + BDNF.) n = number of neurons: EV n = 31, BDNF + EV n = 30, BDNF + Wif1 n = 29, BDNF + Sfrp1 n = 25, BDNF + Fzd8CRD n = 25, BDNF + Dvl1ΔPDZ n = 30. Scale bar: 5 μm.

Article Snippet: pCMV-BDNF was constructed by inserting the open reading frame for murine BDNF into the pEGFP-N1 backbone (Clontech, Mountain View, CA). pBR22-Wif1 expresses murine Wif1 and was a generous gift from Dr. Ken Iwatsuki (Ajinomoto Co., Tokyo, Japan). pCS2 +-Sfrp1 (Addgene plasmid 16693, Cambridge, MA) expresses murine Sfrp1 (Randall Moon Lab). pRK5-mFzd8CRD-IgG (Addgene plasmid 16689) expresses a fusion protein consisting of the extracellular domain of the murine Frizzled-8 protein fused with the human immunoglobulin heavy chain ( ). pCS2 +-mDvl1ΔPDZ-HA expresses a C-terminally HA tagged murine Dishevelled-1 with a deletion of amino acids 276–336 ( ). pTRE-tight-BDNF and pTRE-tight-Wnt2 were constructed by inserting the ORFs for murine BDNF and murine Wnt2, respectively, into the pTRE-tight backbone (Clontech). pTRE-tight-EGFP was constructed by inserting the EGFP ORF from pEGFP-N1 (Clontech) into the pTRE-tight backbone. pCMV-rtTA was constructed by inserting the ORF for the reverse-tet-Transactivator protein from pTRIPZ (Thermo Scientific) into the pEGFP-N1 backbone from which the EGFP coding region was deleted.

Techniques: Expressing

Journal: Molecular and Cellular Neurosciences

Article Title: Neurotrophin and Wnt signaling cooperatively regulate dendritic spine formation ☆

doi: 10.1016/j.mcn.2013.04.006

Figure Lengend Snippet: Transfection with Wnt inhibitors does not prevent BDNF-induced c-Fos expression in cortical neurons. (A) GFP fluorescence, c-Fos immunoreactivity and DAPI fluorescence of representative cell bodies of cortical neurons expressing EV, Wif1, Sfrp1, Fzd8CRD and Dvl1ΔPDZ either alone, or in combination with BDNF. (B) Quantification of normalized nuclear c-Fos immunoreactivity per neuron for each treatment. *** p < 0.001. n = 10 neurons for each treatment.

Article Snippet: pCMV-BDNF was constructed by inserting the open reading frame for murine BDNF into the pEGFP-N1 backbone (Clontech, Mountain View, CA). pBR22-Wif1 expresses murine Wif1 and was a generous gift from Dr. Ken Iwatsuki (Ajinomoto Co., Tokyo, Japan). pCS2 +-Sfrp1 (Addgene plasmid 16693, Cambridge, MA) expresses murine Sfrp1 (Randall Moon Lab). pRK5-mFzd8CRD-IgG (Addgene plasmid 16689) expresses a fusion protein consisting of the extracellular domain of the murine Frizzled-8 protein fused with the human immunoglobulin heavy chain ( ). pCS2 +-mDvl1ΔPDZ-HA expresses a C-terminally HA tagged murine Dishevelled-1 with a deletion of amino acids 276–336 ( ). pTRE-tight-BDNF and pTRE-tight-Wnt2 were constructed by inserting the ORFs for murine BDNF and murine Wnt2, respectively, into the pTRE-tight backbone (Clontech). pTRE-tight-EGFP was constructed by inserting the EGFP ORF from pEGFP-N1 (Clontech) into the pTRE-tight backbone. pCMV-rtTA was constructed by inserting the ORF for the reverse-tet-Transactivator protein from pTRIPZ (Thermo Scientific) into the pEGFP-N1 backbone from which the EGFP coding region was deleted.

Techniques: Transfection, Expressing, Fluorescence