Journal: bioRxiv

Article Title: Small molecule-directed differentiation of submerged-cultured human nasal airway epithelia for respiratory disease modelling

doi: 10.1101/2024.10.17.618953

Figure Lengend Snippet: a) Graphic illustration showing the workflow of generating airway organoids from epithelial fragments of S-diff HNEC, and application in forskolin-induced swelling assays. b) Representative brightfield images of S-diff monolayer derived cystic airway organoids formed after 48 hours. c) Immunofluorescent (top) and brightfield (bottom) images of S-diff HNEC-derived organoids of β-tubulin IV (yellow, left top), MUC5AC (purple, middle top), p63 (red, right top), and DAPI (cyan). d) Representative images of forskolin-induced swelling determined with calcein green AM esters–stained healthy control (HC) and cystic fibrosis (CF; F508del/F508del) organoids, with images taken before stimulation with forskolin (t=0, left panels) and 60 minutes after stimulation (t=60 min, right panels). e,f) Comparison of FIS between HC and CF airway organoids derived from S-diff HNEC (both n=3 independent donors) that were unstimulated or stimulated with forskolin (Fsk). g) CF F508del homozygous organoids derived from S-diff HNEC (n=3 independent donors) were pre-treated with vehicle or CFTR correctors, VX-809, or VX-661/VX-445 for 48 hours. Afterwards FIS was determined following acute stimulation with Fsk, VX-770, or vehicle. h) CF F508del/S1251N airway organoids generated from S-diff HNEC (n=3 independent donors) were used in FIS assays, together with acute stimulation with the CFTR potentiator VX-770. FIS results are depicted as (e) the percentage change in surface area relative to t = 0 (normalized area) measured at 15-min time intervals for 60 minutes, or (f) as area-under-the-curve (AUC) plots (t = 60 min). Data are presented as mean ± SD, and individual data point. Statistical significance was tested using a (f) two tailed paired t-test, (g,h) Tukey’s multiple comparison test. ns = non-significant, *: p<0.05, **: p<0.01. ****: p<0.0001.

Article Snippet: CF airway organoids were pre-treated with CFTR correctors: 5 μM VX-809 (Cat#S1565, Selleck Chemicals), 5 μM VX-661 (Cat#S7059, Selleck Chemicals), 5μM VX-445 (Cat#HY-111772, MedChemExpress), or vehicle control for 48 h. CFTR-dependent organoid swelling was measured after stimulation with forskolin (5 μM, Sigma-Aldrich, Cat#F3917-10mg) and the CFTR potentiators VX-770 (5 μM, Selleck Chemicals, Cat#S1144).

Techniques: Derivative Assay, Staining, Control, Comparison, Generated, Two Tailed Test

Journal: iScience

Article Title: Global functional genomics reveals GRK5 as a cystic fibrosis therapeutic target synergistic with current modulators.

doi: 10.1016/j.isci.2025.111942

Figure Lengend Snippet: Figure 8. Effect of 9j on CFTR function in primary CF human bronchial epithelial (pHBE) cells (A) Representative Ussing chamber recordings of chloride transport measured as transepithelial voltage (Vte).Vte and transepithelial resistance (Rte) were re- corded and used to calculate the equivalent short-circuit current (Isc-eq) using Ohm’s law: Isc-eq = Vte/Rte, as previously reported.49 Fully differentiated pHBE cells homozygous for p.Phe508del were pre-treated with a combination of 3 mM VX445 and 5 mM 661 for 48 h and additionally with DMSO (vehicle) or 500 nM 9j in the last 24 h of assay. Amiloride (ENaC channel inhibitor) was added apically, followed by a combination of IBMX and Fsk (cAMP-elevating agents), VX-770 (CFTR potentiator), and Inh172 (CFTRinh-172, CFTR specific inhibitor). (B) Summary of calculated equivalent short-circuit currents (Isc-eq) induced by IBMX/Fsk + VX-770 and inhibited by Inh172. Values are represented as mean ± SD (n = 7 biological replicates). * represents significance (p value <0.05, unpaired t test).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-FLAG (clone M2) Sigma-Aldrich Cat#F1804; RRID: AB_262044 Donkey anti-mouse Alexa Fluor 647 Invitrogen Cat#A31571; RRID: AB_162542 Mouse monoclonal anti-CFTR CFF Therapeutics Cat#596; RRID: AB_2923486 Mouse monoclonal anti-calnexin (clone 37) BD Transduction Laboratories Cat#610523; RRID: AB_397883 Chemicals, peptides, and recombinant proteins Puromycin Sigma-Aldrich Cat#P8833 Blasticidin InvivoGen Cat#ant-bl G418 Sigma-Aldrich Cat#A1720 Doxycycline Sigma-Aldrich Cat#9891 Forskolin Sigma-Aldrich Cat#F6886 IBMX Sigma-Aldrich Cat# I5879 CFTRinh-172 MedChemExpress Cat#HY-16671 Genistein Sigma-Aldrich Cat#G6649 VX-445 Selleckchem Cat#S8851 VX-661 Selleckchem Cat#S7059 VX-770 Selleckchem Cat#S1144 VX-809 Selleckchem Cat#S1565 Hoechst 33342 Sigma Aldrich Cat#B2261 9g (CCG-273463) This work, synthesized as in Rowlands et al.45 N/A 9j (CCG-273441) MedChemExpress Cat#HY-47573 Resazurin Sigma-Aldrich Cat#R7017 Critical commercial assays SsoFast EvaGreen Bio-Rad Cat#1725201 Deposited data Image and data analysis of the primary and secondary siRNA screens This paper https://doi.org/10.5281/zenodo.6617740 CFTR interactomes This paper plus Pankow et al.,28 Canato et al.,29 Reilly et al.,30 Tomati et al.,31 Dang et al.,32 Almaça et al.,33 Simpson et al.34 https://doi.org/10.5281/zenodo.6646236 Experimental models: Cell lines CFBE41o- mCherry-Flag-wt-CFTR Botelho et al.26 https://doi.org/10.1038/srep09038 CFBE41o- mCherry-Flag-p.Phe508del-CFTR Botelho et al.26 https://doi.org/10.1038/srep09038 CFBE41o- mCherry-Flag-p.Phe508del-4RK Canato et al.29 https://doi.org/10.1007/s00018-018-2896-7 CFBE41o- mCherry-Flag-p.Phe508delp.Arg1070Trp-CFTR Canato et al.29 https://doi.org/10.1007/s00018-018-2896-7 CFBE41o- mCherry-Flag-p.Phe508delp.Gly550Glu-CFTR Canato et al.29 https://doi.org/10.1007/s00018-018-2896-7 CFBE41o- mCherry-Flag- DD/AA-CFTR Canato et al.29 https://doi.org/10.1007/s00018-018-2896-7 CFBE41o- YFP-H148Q/I152L wt-CFTR Laboratory of Nicoletta Pedemonte https://doi.org/10.1152/ajpcell.00507.2010 CFBE41o- YFP-H148Q/I152L p.Phe508del-CFTR Laboratory of Nicoletta Pedemonte https://doi.org/10.1152/ajpcell.00507.2010 CFBE41o- wt-CFTR Laboratory of John P. Clancy https://doi.org/10.1113/jphysiol.2005.096669 CFBE41o- p.Phe508del-CFTR Laboratory of John P. Clancy https://doi.org/10.1113/jphysiol.2005.096669 (Continued on next page) e1 iScience 28, 111942, March 21, 2025

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