A Novel High Throughput High Content Analysis Assay for Intermediate Filament Perturbing Drugs
Keratins form the intermediate filament (IF) component of the epithelial cytoskeleton. Depolymerisation of these filaments causes the cell to collapse and become more plastic. We have previously shown that short chain fatty acids may trigger depolymerisation of keratins through altered protein acetylation. Currently, there is no single functional assay for screening of the cytoskeleton. The aim of this study is to develop a high-throughput assay to quantify IF depolymerisation and to apply as a screen for IF-perturbing nutrients and drugs. Three treatments were used in a proof-of-principle study: the anti-fungal drugs griseofulvin and cordycepin (the former a c-mitotic drug known to suppress microtubule growth, the latter inducing abnormal mitosis by suppressing microtubule dynamics) and sodium butyrate, a histone deacetylase inhibitor which disrupts IF formation in cancer cells via post-translational modification of keratins.
Methods were optimised for cell fixation using methanol or formalin, permeabilising agents for Keratin 8 (krt8) antibody (triton-x100, digitonin and saponin) and blocking of non-specific binding prior to cell staining using BSA. High Content Analysis (HCA) was employed to quantify cell staining intensities by comparing co-occurrence of adjacent pixel intensities. Immunocytochemistry was used to identify Krt 8 intermediate filaments. Indicators of depolymerisation include Krt 8 fluorescence intensity, filamentousness or texture intensity, fibre spot count and fibre spot total area. All treatments resulted in significant decreases for texture intensity. Assessment of this assay using a Z prime calculation recorded gace a statistic of 0.95, indicating suitability for high-throughput applications.
In conclusion, a HCA assay for intermediate filament integrity has been demonstrated, establishing proof of principle with griseofulvin, and cross-validating with two further treatments assayable using this method.