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