Phosphorylation state and protein levels measured by imaging in BRAF(V600E/D) melanoma cell lines following treatment with combinations of two compounds (immunofluorescence dataset 1 of 4) - Dataset (ID:20274)

HMS Dataset ID: 20274
Dataset Title: Phosphorylation state and protein levels measured by imaging in BRAF(V600E/D) melanoma cell lines following treatment with combinations of two compounds (immunofluorescence dataset 1 of 4)
Publication(s) Using Dataset: PMID: 28069687
Project Summary Page(s): lincs.hms.harvard.edu/fallahi-sichani-molsystbiol-2017
Screening Lab Investigator: Mohammad Fallahi-Sichani
Screening Principal Investigator: Peter K. Sorger
Assay Description: Three human melanoma cell lines were treated in 2 replicates with Vemurafenib at 3 doses in combination with DMSO or 2 doses of 9 other compounds. This dataset presents the mean intensity measurements for 6 protein or phosphoprotein readouts (KI-67, Rb (pS807; pS811), NGFR, S6 (pS240; pS244), c-Jun, and c-Jun (pS73)) extracted from microscopy images across each condition.
Assay Protocol: 1. Cells were seeded at the following densities in 96-well plates (Corning, cat. #3603) and grown in full growth media: WM-115, 10,000 cells per well; COLO 858, 5,000 cells per well; and A-375, 4,000 cells per well. Cell counts were based on measurements using a Cellometer Auto T4 Cell Viability Counter (Nexcelom Bioscience).
2. After 24 hr of normal growth, cells were treated in duplicates using a Hewlett-Packard (HP) D300 Digital Dispenser with Vemurafenib at 0, 0.32 and 1 μM in combination with DMSO or 2 doses of Trametinib (0.2, 0.6 μM), Defactinib (1, 3 μM), PF562271 (1, 3 μM), Dasatinib (1, 3 μM), Saracatinib (1, 3 μM), JNK-IN-8 (1, 3 μM), AZD8055 (0.2, 0.6 μM), Pictilisib (1, 3 μM), or Palbociclib (1, 3 μM) for 48 hr.
3. Cells were fixed in 4% paraformaldehyde for 20 min at room temperature, washed with PBS with 0.1% Tween 20 (Sigma-Aldrich) (PBS-T), permeabilized in methanol for 10 min at room temperature, rewashed with PBS-T, and blocked in Odyssey Blocking Buffer for 1 hr at room temperature.
4. Cells were incubated overnight at 4°C with primary antibodies in Odyssey Blocking Buffer. The following primary antibodies were used at the specified dilutions: anti-S6 (pS240; pS244), 1:800; anti-c-Jun, 1:800; anti-c-Jun (pS73), 1:800; anti-KI-67, 1:400; anti-NGFR, 1:1600; and anti-Rb (pS807; pS811), 1:400.
5. Following treatment with primary antibodies, cells were stained with donkey anti-rabbit, anti-mouse or anti-goat IgG secondary antibodies labeled with Alexa Fluor 647, Alexa Fluor 488, and Alexa Fluor 568, respectively, that had been diluted 1:2000 in Odyssey Blocking Buffer. Cells were washed once in PBS-T and once in PBS and were then incubated in 250 ng/mL Hoechst 33342 and 1:800 Whole Cell Stain (blue; Thermo Scientific) solutions for 20 min.
6. Cells were washed twice with PBS and imaged with a 10x objective on an Operetta imaging system (PerkinElmer). Nine to 11 sites were imaged in each well.
7. Image segmentation, analysis and signal intensity quantification were performed using Acapella software (PerkinElmer). Hoechst 33342 and whole cell staining were used for image segmentation. The Alexa Fluor 488, 568 and 647 signal intensity data for the proteins of interest are presented as the mean intensity of each signal averaged across the 9 to 11 sites imaged in each well.
HMS Dataset Type: Microscopy/Imaging
Date Publicly Available: 2016-12-22
Most Recent Update: 2017-01-20