Phosphorylation state and protein levels measured in BRAF(V600E/D) melanoma cell lines monitored by imaging - Dataset (ID:20219)
|HMS Dataset ID:||20219|
|Dataset Title:||Phosphorylation state and protein levels measured in BRAF(V600E/D) melanoma cell lines monitored by imaging|
|Publication(s) Using Dataset:||PMID: 25814555|
|Project Summary Page(s):||lincs.hms.harvard.edu/fallahi-sichani-molsystbiol-2015|
|Screening Lab Investigator:||Mohammad Fallahi-Sichani|
|Screening Principal Investigator:||Peter Sorger|
|Assay Description:||This is the numerical data extracted from microscopy images measuring the mean intensity for 7 signals in multiple replicates across 4 cell lines and 3 treatment conditions (10 drug doses). Each data row reports the mean intensity of three fluorescent channels (Alexa 488, Alexa 568 and Alexa 647) for each measured protein (or phospho protein). Cells were seeded at the following densities in 96-well plates in full growth media for 24 hr: WM115 (10,000 cells per well), WM1552C (7,000 cells per well), COLO858 and LOXIMVI (5,000 cells per well). Cells were then treated in duplicates using Hewlett-Packard (HP) D300 Digital Dispenser with nine doses of vemurafenib, JNK-IN-8 or their combination for 24 hr. When combined, drugs were added at a 1:1 ratio at indicated doses. Several related HMS datasets from the same project also are available: experimental data in #20217 and #20218 and signature data in #20229, #20230, and #20231.|
1. Cells were seeded at the following densities in 96-well plates (Corning) and grown in full growth media: WM115 (10,000 cells per well), WM1552C (7,000 cells per well), COLO858 and LOXIMVI (5,000 cells per well).|
2. After 24 hr of normal growth, cells were treated in duplicates using Hewlett-Packard (HP) D300 Digital Dispenser with nine doses of vemurafenib, JNK-IN-8 or their combination for 24 hr. When two drugs were added in combination, the drugs were added at a 1:1 ratio each at the indicated dose.
3. Cells were fixed in 2% paraformaldehyde for 10 min at room temperature and 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 4C with primary antibodies in Odyssey Blocking Buffer.
5. The following primary antibodies with specified animal sources and catalog numbers were purchased from Cell Signaling Technology and were used in specified dilution ratios: p-S6(S235/S236) (rabbit mAb; 1:800; 4858), p-ERK(T202/Y204) (rabbit mAb; 1:800; 4370), p-c-Jun(S73) (rabbit mAb; 1:800; 3270), Ki-67 (mouse mAb; 1:400; 9449), p-Histone H3 (S10) (mouse mAb; 1:800; 9706). A goat polyclonal antibody to p-Rb(S807/S811) (1:400; sc-16670) was purchased from Santa Cruz Biotechnology. A mouse monoclonal antibody to p21 Kip1 (1:200; BD-610241) was purchased from BD Biosciences.
6. Following treatment with primary antibodies, cells were stained with rabbit, mouse or goat secondary antibodies labeled with Alexa Fluor 647, Alexa Fluor 488 and Alexa Fluor 568 (Invitrogen). Cells were washed once in PBS-T, 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.
7. Cells were then washed twice with PBS and imaged with a 10X objective on Operetta. Nine sites were imaged in each well. Image segmentation, analysis and signal intensity quantification were performed using Acapella software. Hoechst and whole cell staining were used for image segmentation. The Alexa Fluor 488, 568, and 647 data for the proteins of interest are presented as the mean intensity of each signal averaged across the nine sites imaged in each well.
|HMS Dataset Type:||Microscopy/Imaging|
|Date Publicly Available:||2015-01-23|
|Most Recent Update:||2016-10-28|