br A cell transfection br A cells were
A375 cell transfection
A375 cells were seeded at the density of 0.15 million per well in 6-well plates. At 24 h after seeding, transfection was performed using 2 mg plasmid and 4 mL of X-treme GENE HP reagent (06 366 236 001, Roche) per well with the standard protocol provided by the manufacturer.
Kinase inhibition with small molecules
BRAFV600E inhibitor vemurafenib and MEK inhibitor CI1040 (stock solutions of 10 mM/mL in DMSO, Selleckchem) were added to the cells 18 h after transfection independently or in combination at final concentrations of 1 mM and 10 mM, respectively. The same vol-ume of DMSO was added to the control samples. Cells were treated either for 3 h or for 2 days. At the end of the treatment, cells were labeled with IdU during 20-minute incubation and subsequently harvested by 5-minute TrypLE digestion and 10-minute PFA cross-linking as described above.
Crosslinked cells were washed twice with cell staining media (CSM, PBS with 0.5% BSA). After centrifugation, ice-cold methanol was used to resuspend the cells, followed by 10-minute permeabilization on ice or for long-term storage at 80 C.
The MaxPAR antibody conjugation kit (Fluidigm) was used to generate isotope-labeled Resveratrol using the manufacturer’s standard protocol. After conjugation, the antibody yield was determined based on absorbance of 280 nm. Candor PBS Antibody Stabilization solution (Candor Bioscience GmbH) was used to dilute antibodies for long-term storage at 4 C.
Barcoding and staining protocol
Formalin-crosslinked and methanol-permeabilized cells were washed three times with CSM and once with PBS. Cells were incu-bated in PBS containing barcoding reagents of 89Y (100 nM), 103Rh (2 mM), 105Pd (100 nM), 106Pd (100 nM), 108Pd (100 nM), 110Pd (100 nM), 113In (200 nM), 115In (100 nM), and 209Bi (20 nM) for 30 min at room temperature and then washed three times with CSM. Barcoded cells were then pooled and stained with the metal-conjugated antibody mix (Table S2) at room temperature for 1 h. The antibody mix was removed by washing cells three times with CSM and once with PBS. For DNA staining, iridium-containing intercalator (Fluidigm) diluted in PBS with 1.6% PFA was incubated with the cells at 4 C overnight. On the day of the measurement, the intercalator solution was removed, and cells were washed with CSM, PBS, and ddH2O. After the last washing step, cells were resuspended in ddH2O and filtered through a 70-mm strainer.
Mass cytometry analysis
EQ Four Element Calibration Beads (Fluidigm) were added to cell suspensions in a 1:10 ratio (v/v). Samples were analyzed on a CyTOF2 (Fluidigm). The manufacturer’s standard operation procedures were used for acquisition at a cell rate of 500 cells per second. After the acquisition, all FCS files from the same barcoded sample were concatenated (Bodenmiller et al., 2012). Data were then normalized, and bead events were removed (Finck et al., 2013) before doublet removal and de-barcoding of cells into their corresponding wells using a doublet-filtering scheme and single-cell deconvolution algorithm (Zunder et al., 2015). Subsequently, data were processed using Cytobank (https://www.cytobank.org). Additional gating on the DNA channels (191Ir and 193Ir) and 139La/141Pr was used to remove remained doublets, debris and contaminating particulate.
QUANTIFICATION AND STATISTICAL ANALYSIS
Data preprocessing and BP-R2 analysis Data preprocessing
Raw data were transformed using the inverse hyperbolic sine transform with a cofactor of 5:
data = arcsinhðdataraw=5Þ
Except where use of raw data values is specifically noted, all visualizations and analyses were performed using transformed data. Data binning
For data binning, the range between the lower and upper 2.5% of observations was divided into ten equal bins bin1,...,bin10. The observations in the lower and upper 2.5% were assigned to the lowest and highest bins, respectively. In order to be able to compare expression levels between samples within a time course experiment, all observations of the time course were pooled to determine the binning.