Figure 2
From: Identification of Novel Inhibitors of DLK Palmitoylation and Signaling by High Content Screening
A High Content Imaging screen identifies ketoconazole as the most potent compound to inhibit DLK-GFP puncta formation. (A) HEK293T cells cotransfected with DLK-GFP plus mCherry-NLS were treated with 2BP or vehicle and fixed to detect GFP, mCherry and the nuclear marker DAPI. 2BP reduces DLK-GFP puncta without affecting mCherry-NLS expression or DAPI signal. Scale bar: 25 µm. (B) HEK293T cells were seeded into 12 wells of a 96-well plate and transfected with DLK-GFP and NLS-mCh cDNA and then treated with 2BP (20 μM in DMSO) or 0.1% (v/v) DMSO vehicle as in A. Cells were fixed and imaged using an ImageXpress High Content Imaging system to detect GFP and NLS-mCh signals. Assay quality was determined by calculating the z-prime (z′) for 6 determinations per condition (z′ = S/R, S = [(Mean of Vehicle treated – 3xSD)-(Mean of 2BP – 3xSD)], R = Vehicle Mean – 2BP mean). (C) Design of the high-throughput screen for compounds that inhibit DLK-GFP punctate localization. (D) The effect of 1200 compounds from the Prestwick Chemical Library™ on DLK puncta per transfected cell (mean of 2 determinations per compound) was calculated using ImageXpress Image Analysis ‘TransFluor’ and Multi-Wavelength Scoring (MWS) modules. Compounds that decreased the number of transfected cells (from mCherry-NLS count) or the total number of cells (from DAPI count) by greater than 30%, relative to the mean of vehicle treated controls, are not plotted due to likely cytotoxicity or non-specific effects. Red and blue lines indicate 3 standard deviations (3 SD) above and below the mean of all determinations, respectively. Compounds that decreased DLK puncta per transfected cell below this 3 SD cut-off were considered ‘Hits’. The most potent ‘hit’, ketoconazole, is highlighted in red.
