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High content, high-throughput screening for small molecule inducers of NF-κB translocation

PLoS ONE (2018) 13(6)
DOI: 10.1371/journal.pone.0199966
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Abstract

NF-κB is an important mediator of immune activity and its activation is essential in mounting immune response to pathogens. Here, we describe the optimization and implementation of a high-throughput screening platform that utilizes high content imaging and analysis to monitor NF-κB nuclear translocation. We screened 38,991 compounds from three different small molecule libraries and identified 103 compound as hits; 31% of these were active in a dose response assay. Several of the molecules lacked cytotoxicity or had a selectivity index of more than 2-fold. Our image-based approach provides an important first step towards identifying small molecules with immunomodulatory activity.

Figures

  • Fig 1. Schematic representation of NF-kB nuclear translocation assay.
  • Fig 2. Optimizing activation stimuli for NF-κB nuclear translocation. HUVECs were seeded overnight, stimulated with TNF-α for 30 min and stained for NF-κB. (A) Images from the DAPI and FITC channel. White arrows indicate non-stained nuclei in FITC channel; green arrows showing stained nuclei in FITC channel (B) Time course of translocation. HUVECs were stimulated with 10 ng/mL TNFα. (C) Translocation in response to different inducers. HUVECs were stimulated for 30 min with 10 ng/mL TNF-α, 100 ng/mL lipopolysaccharide (LPS), 10 μM phorbol myristate acetate (PMA), 10 μM prostratin, or 10 μM calcimycin (D) Nuclear translocation in response to TNF-α. HUVECs were stimulated for 30 min. The average pixel intensity was measured in both the nucleus and cytoplasm of each cell and a translocation value obtained calculating the NUC/CYT. Results are shown in boxes and whiskers. Boxes denote the interquartile range with the median represented by line inside the boxes. Whiskers show maximum and minimum values. Differences between conditions tested was assessed using the Kruskal- Wallis test. Data are a representative of two different experiments (n = 2) with at least 8 replicates per condition tested.
  • Fig 3. Antibody optimization. HUVECs were seeded overnight, stimulated with 100 ng/mL TNF-α and stained for NF-κB p65. NF-κB p65 average pixel intensity was measured in both the nucleus and cytoplasm of each cell and a translocation value obtained by calculating the NUC/CYT (A) Cells stained with mouse monoclonal, rabbit monoclonal and rabbit polyclonal NF-κB p65 primary antibodies. (B) Superimposed images from DAPI (nuclei) in blue and FITC (NF-κB p65) in green in TNF- α treated and DMSO control cells. Cyan stained cells are co-labelled (C) Optimization of antibody concentrations; different concentrations of primary and secondary antibodies were tested. Green circles—TNF-α stimulated cells; black squares–DMSO control. Data are a representative of two different experiments (n = 2) with at least 14 replicates per condition tested.
  • Fig 4. Image analysis pipeline. (A) Low and high pixel intensity threshold setting in DAPI channel to identify cells. Uneven shapes and features created with high threshold setting. Uniform features created with low threshold intensity. (B) Stepwise pipeline used to create nuclei, whole cell and cytoplasm masks. Fill holes operation done from threshold settings to create even features. Nuclei mask created from fill holes operation by shrinking features by a number of pixel intensities and size. Extended nuclei mask created from nuclei mask by increasing features by a few pixel intensities. Whole cell mask created from nuclei mask by increasing features be a number of pixel intensities. Cytoplasm mask created from both extended nuclei mask and whole cell mask (C) Different cell compartments identified using the pipeline. Yellow, blue and light green representing nuclei, cytoplasm and extended nuclei respectively.
  • Fig 5. Screen validation. HUVECs were seeded overnight, stimulated with 100 ng/mL TNF-α and stained for NF-κB p65. NF-κB p65 average pixel intensity was measured in both the nucleus and cytoplasm of each cell and a translocation value obtained by calculating the NUC/CYT. Screen validation was performed as three independent experiments, each with duplicate plates of negative control (DMSO), positive control (100 ng/mL TNF-α) and dose response (10-point, 3-fold dilutions of TNF-α). (A-C) are data from 1 plate of each run for positive and negative controls. (A) Run 1 (B) Run 2 (C) Run 3. (D) Dose response curves from all three days. NUC/CYT was normalized and plotted using non- linear 4-parameter fit in order to calculate the EC50 = effective concentration 50%, defined as the concentration at which 50% of the maximum response is seen.
  • Fig 6. Small molecule library screen. (A) Plate lay out for single point compound screen at 10 μM. Positive control: 100 ng/mL TNF-α in columns 1 and 24. Negative control: 1% DMSO in columns 2 and 23. Screening data from (B) MyriaScreen II. (C) TimTec. (D) ChemBridge libraries. Red line denotes cutoff for hit identification (NUC/CYT 1.3).
  • Table 1. Hits from NF-κB nuclear translocation assay.
  • Fig 7. Confirmed hits and structures: Small molecules confirmed as hits in dose-response experiment with their corresponding structures.

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CITATION STYLE

APA

Njikan, S., Manning, A. J., Ovechkina, Y., Awasthi, D., & Parish, T. (2018). High content, high-throughput screening for small molecule inducers of NF-κB translocation. PLoS ONE, 13(6). https://doi.org/10.1371/journal.pone.0199966

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