Biology subjects

Culley, J.

Publications and source records attributed to Culley, J..

3 recordsLinked to original sources

bioRxiv2026

Multi-Site Reproducibility Study of 3D High-Content Analysis with Dual-View Oblique Plane Microscopy

High content imaging is being applied to achieve quantitative fluorescence readouts in increasingly complex 3-dimensional (3D) cell culture models such as spheroids and organoids. Compared to conventional 2D assays, 3D assays better represent biological heterogeneity but require more complex sample preparation, 3D imaging and 3D image analysis that can affect the accuracy and precision of such assays. We used spheroids formed from the NRAS-activated melanoma cell line 19161 modified to express an ERK kinase translocation reporter (KTR) as an exemplar 3D phenotypic assay carried out in 96-well plates. The spheroids were treated with the ERK activator TPA and a range of concentrations of the MEK inhibitor Binimetinib. 3D live-cell imaging with sub-cellular spatial resolution was performed using a dual-view oblique plane microscope (dOPM) - a form of single-objective light-sheet microscope - and the experiment was performed separately at 4 different institutes. The results were analysed using an identical 3D analysis pipeline and parameters. We assessed the variation in assay readout using a linear mixed effects model. Random variance at the well level was negligible (SD = 0.0048 relative to range of KTR biosensor readout at reference site of 0.17), indicating low technical noise. Treatment effects were dose-dependent and highly statistically significant compared to DMSO control across all sites (Dunnett-corrected p < 0.001). The range in KTR readout between the minimum (3.5 M Binimetinib) and maximum (100 nM TPA) treatments varied between 59 to 96% relative to the reference site. Measured bias in KTR readout between sites was between 6 and 12% of the range of the reference site. This study quantifies the reproducibility of a 3D live spheroid-based assay employing a fluorescence biosensor requiring readout out at the per-cell level using the dOPM platform and discusses areas where experimental protocol could be improved in the future to further improve reproducibility.

bioengineering↗

bioRxiv2021

Characterisation of a nucleo-adhesome

In addition to central functions in cell adhesion signalling, integrin-associated proteins have wider roles at sites distal to adhesion receptors. In experimentally defined adhesomes, we noticed that there is clear enrichment of proteins that localise to the nucleus, and conversely, we now report that nuclear proteomes contain a class of adhesome components that localise to the nucleus. We here defined a nucleo-adhesome, providing experimental evidence for a remarkable scale of nuclear localisation of adhesion proteins, establishing a framework for interrogating nuclear adhesion protein functions. In adding to nuclear FAKs known roles in regulating transcription, we now show that nuclear FAK regulates expression of many adhesion-related proteins that localise to the nucleus and that nuclear FAK binds to the adhesome component and nuclear protein Hic-5. FAK and Hic-C work together in the nucleus, co-regulating a subset of genes transcriptionally. We describe the first nucleo-adhesome using a squamous cancer cell model, and demonstrate the new principle that there are nuclear adhesion protein subcomplexes that cooperate to control transcription. O_FIG O_LINKSMALLFIG WIDTH=177 HEIGHT=200 SRC="FIGDIR/small/458428v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@1c841ceorg.highwire.dtl.DTLVardef@586a75org.highwire.dtl.DTLVardef@b54439org.highwire.dtl.DTLVardef@921537_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2020

Chemical-Genetic Interrogation of Nuclear Size Control Reveals Cancer-Specific Effects on Cell Migration and Invasion

Lower survival rates for many cancer types correlate with increases or decreases in nuclear size/scaling in a tumor-type/tissue-specific manner. Postulating that nuclear size changes confer a fitness advantage on tumor cells, we screened for FDA/EMA-approved compounds that reverse tumor nuclear size changes in cell lines from three such tumor types: prostate adenocarcinoma, colonic adenocarcinoma, and small-cell squamous lung cancer. We found distinct, largely non-overlapping sets of compounds that either rectify or exacerbate nuclear size changes for each tumor type. Nuclear size phenotypes across cell lines clustered particular classes of compounds including serotonin uptake inhibitors, cyclo-oxygenase inhibitors, beta-adrenergic receptor agonists, monoamine oxidase inhibitors, and Na+/K+ ATPase inhibitors. Nearly all compounds selected for further investigation inhibited cell migration and/or invasion, suggesting that targeting nuclear size control pathways in chemotherapy regimens could improve patient survival.

cancer biology↗