CFTR Interactome Mapping Using the Mammalian Membrane Two-Hybrid High-Throughput Screening System (2022)
Figure 6

Figure 6 - Effects of lentivirus-mediated shRNA knockdown of the FGL2 and CFTR genes in intestinal organoids.

A) Schematic of patient-derived intestinal organoid acquisition and expansion followed by two functional assays. Created with BioRender.com.

B) qPCR Validation of lentivirus-mediated shRNA knockdown of FGL2 and CFTR in intestinal organoids. Significance of sample differences was calculated using the one-tailed student t-test (n=2 biological replicates). All values are mean ± s.d. EV = empty vector.

C) Representative confocal microscopy images of calcein green labelled FIS organoids of a healthy individual and a CF patient, before and 60 min after stimulation with Fsk (5 uM). Scale bar, 200 μm.

D) Quantification of FIS response in wt-CFTR organoids (ORG-01(HC)) shown in (C) as area under the curve at 60 min. Significance of sample differences was calculated using two-way ANOVA (n=2 biological replicates). All values are mean ± s.d.

E) Quantification of FIS response in F508del-CFTR organoids (ORG-BX-002 (dF/dF)) shown in (C) as area under the curve at 60 min. Significance of sample differences was calculated using two-way ANOVA (n=2 biological replicates). All values are mean ± s.d.

F) Tracing of Ussing chamber measurements of the transepithelial current produced by 2D monolayer cultures of specified organoids. The F508del-homozygous monolayers with the knockdowns have been rescued with Trikafta.

Data Availability

The data produced in this study are available in the following databases:

MaMTH-HTS Interaction Data: IID Integrated Interaction Database (http://ophid.utoronto.ca/iid). Part of the IMEx Consortium, (Orchard et al, 2012; Porras et al, 2020). Deposited as MaMTH-HTS-2021, identifiable with PMID.

Acknowledgements

This research was supported by funding from the Cystic Fibrosis Canada Basic Science Research Grant (#72046178), the Canadian Cancer Society Research Institute (#703889), Genome Canada via Ontario Genomics (#9427 & #9428), Ontario Research fund (ORF/DIG-501411 & RE08-009), CQDM (Quantum Leap) and Brain Canada (Quantum Leap), and Cancer Research Society (#23235), Genentech Inc. US (CLL-018879), and Canadian Institutes for Health Research (#420989) to Igor Stagljar.

Furthermore, this work was supported by UIDB/04046/2020 and UIDP/04046/2020 centre grants from FCT-Fundação para a Ciência e a Tecnologia (FCT), Portugal (to BioISI). High-content screening was performed at Faculty of Sciences of the University of Lisboa Microscopy Facility, a node of the Portuguese Platform of BioImaging (PPBI), FCT reference PPBI-POCI-01-0145-FEDER-022122. MCP is recipient of a fellowship (from SFRH/PD/BD/114393/2016) BioSys PhD programme (PD/00065/2012), from FCT.

Computational analyses were supported in part by funding from Ontario Research Fund (RDI #34876), Natural Sciences Research Council (NSERC #203475), Canada Foundation for Innovation (CFI #225404, #30865), IBM and Ian Lawson van Toch Fund.

M.B. is a CIHR New Investigator (MSH-130178) and a Chancellor's Research Chair. This work was supported by grants from the Canadian Institutes of Health Research (FDN-154318) and CFI innovation 2020 fund to M.B.

wt- and F508del-CFTR cDNA entry constructs (Ref Seq: NM_000492.3) used in cloning were generously gifted from Dr. Johanna Rommens.

Author Contributions

SHL: performed experiments, analyzed data and wrote the manuscript.

JS: performed experiments, analyzed data and wrote the manuscript.

LBS: performed experiments and analyzed data.

WI: performed experiments and analyzed data.

JCS: performed experiments and analyzed data.

HMB: supervised research, planned, performed and analyzed experiments. Contributed to final manuscript.

MLP: performed experiments and analyzed.

MCP: performed experiments.

MTM: performed experiments and analyzed data.

MZ: performed experiments.

OL: performed experiments and analyzed data.

ME: performed experiments.

MK: performed data analysis and interactome assembly

AL: performed and assisted with experiments, assisted with data analysis

PT: performed and assisted with experiments

LLV: performed and assisted with experiments

IA: performed and assisted with experiments

FA: performed and assisted with experiments

VW: performed and assisted with experiments

IG: performed and assisted with experiments

TOB: performed and assisted with experiments

ZY: provided assistance with experimental design and implementation

MM: performed experiments

MB: supervised research and contributed to final manuscript.

IJ: performed data analysis and interactome assembly

TG: supervised and assisted with experimental design of organoid research.

CEB: supervised research and assisted with experimental design

MDA: supervised research and contributed to final manuscript.

IS: supervised research and contributed to final manuscript.

Disclosure statement and competing interests

IS is named as a co-inventor on a patent for the MaMTH technology.