Hela (human cervical cancer) and OPM2 (human multiple myeloma) cell lines were obtained from the American Type Culture Collection and maintained in a humidified atmosphere of 37°C in 5% CO2. Hela cells were cultured in Dulbecco’s Modified Eagle
Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS), penicillin (100 units/mL) , and streptomycin (100 µg/mL), while OPM2 cells were maintained in RPMI- 1640 media with the same supplements.
55 2.3.2 FLS Cell Culture
FLS from 6 individuals were collected at the time of synovectomy or total joint replacement. Three were biologic-naïve RA patients which fulfilled the American College of Rheumatology 1997 criteria for RA classification. Three were individuals who were
undergoing surgical resection of tissue for reasons unrelated to rheumatologic disorders. Tissues were stored under liquid N2 until the time of FLS culture. Synovial tissue was
minced and immobilized in a tissue culture plate and covered with Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) and 1% PenStrep Solution (Gibco, Grand Island, NY) and were maintained in a humidified
atmosphere of 37°C in 5% CO2. Media was changed regularly until the FLS expanded to fill
the entire plate. These cell lines from primary FLS were utilized from the third to ninth passage.
2.3.3 Small Interfering RNA transfection in FLS
Akt and scrambled non-targeting siRNA were purchased from Dharmacon Research, Inc. (Lafayette, CO, USA). The FLS cells were seeded in culture plates and grown to 80%
confluence. FLS cells (3 x 105) were transfected with a variety of siRNA concentrations utilizing the Amaxa Human Dermal Fibroblast Nucleofector kit (NHDF-adult) with high efficiency program U-023 for human cells. After transfection, cultures were incubated at 37°C for 12 h and then placed in fresh culture media. Forty eight hours after transfection, the cells were harvested and the Akt protein expression levels were determined by Western blot analysis.
56 2.3.4 Western Blot Analysis
FLS from normal or rheumatic human samples were obtained and cultured. Cells were either not stimulated or stimulated with 100 nM TNFα for 30 min and then lysed in ice cold RIPA buffer plus protease and phosphatase inhibitors (10 mM Tris-Cl (pH 8.0), 1 mM
ethylenediaminetetraacetic acid (EDTA), 0.5 mM ethyleneglycoltetraacetic acid (EGTA), 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 140 mM NaCl, 1X cOmplete ULTRA Protease Inhibitor and 1X PhosSTOP Phosphatase Inhibitor Cocktail (Roche, Indianapolis, IN)). Protein lysates were normalized using a BCA assay (Thermo Scientific, Rockford, IL) and run on a Mini-PROTEAN TGXTM gel (Any kDTM, 15- well comb, 15 µl) (Bio-Rad, Hercules, CA). Between 6-10 µg total protein was run per independent experiment. All lysates for an independent experiment were run on the same blot for direct comparison of signaling strength. Antibodies were from Cell Signaling Technology (Danvers, MA) or Trevigen (Gaithersburg, MD): P-Akt (S473) (D9E) XP® Rabbit mAb), anti-G3PDH/GAPDH, Akt Rabbit Ab, and anti-rabbit IgG, HRP-linked Antibody. All antibodies were diluted in 1x Tris-Buffered Saline with Tween 20 (Cell Signaling, Danvers, MA) + 3% Bovine Serum Albumin (BSA). Images were scanned and imported into ImageJ where images were converted into an 8-bit black and white image.
2.3.5 Cell Loading and Single-Cell Analysis by Capillary Electrophoresis
FLS from normal and rheumatoid arthritis subjects were separately cultured in custom cell chambers as described previously.18 Chambers were placed on the stage of a
custom-built single-cell CE-LIF system (fluorescence excitation at 473 nm, emission at 530 nm).19 Cells were microinjected with 100 µM peptide VI-B (6FAM-GRP-MeArg-AFTF-
57
MeAla-Amide) using a Transjector 5246 microinjection system (Eppendorf AG, Hamburg, Germany) and bathed in a continuous flow of extracellular buffer (ECB; 10 mM HEPES, 135 mM NaCl, 5 mM KCl, 1 mM CaCl2, pH 7.4, 37°C) during incubation and analysis. After
microinjection (5 min), individual cells were rapidly lysed with a 9 ns pulse from a Nd:YAG laser (New Wave Research, Bozeman, MT) and the cellular contents simultaneously electrokinetically injected (5 s at -125 V/cm) into an overlying 30 µm inner diameter (Polymicro Technologies, Phoenix, AZ). Electrophoresis was performed in 100 mM borate, 15 mM SDS, pH 11.6 with a field strength of -250 V/cm. Electropherograms were integrated using customized software written in MATLAB (Natick, MA).17 Detailed information regarding peptide fragment nomenclature and identification is available in supplemental information.
2.3.6 Identification of Peptide Fragmentation Products
Synthetic standards of all possible fluorescent peptide fragments of VI-B were synthesized as described previously.2 Under the electrophoretic conditions used, the phosphorylated peptide was fully resolved from the parent peptide and each potential fragment. Thus, the fragments formed and their identities were identifiable as described previously.2 Each peak present in the individual cells were readily matched by migration time to a fluorescent peptide fragment or the intact or phosphorylated reporter.
2.3.7 Flow Cytometry
Antibodies conjugated with AlexaFluor 647 (AF647) were utilized for flow cytometry. CD120a (Tumor Necrosis Factor Receptor 1)–AF647 and isotype control
58
incubation and data acquisition, cells were suspended in incubation buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, 20 mM 4-(2-hydroxyethyl)-1-
piperzineethanesulfonic acid, 5 mM EDTA, 2% FBS). FLS cells were removed from tissue culture dishes with Accutase (Sigma Aldrich, St. Louis, MO) per manufacturer's instructions. CD120a-AF647 antibody and 500 nM SYTOX Green (ThermoFisher Scientific, Waltham, MA) was added to FLS cells (0.1 µg/mL) and incubated for 30 min in the dark at 25°C. Cells were washed twice with incubation buffer by centrifugation at 300 x g for 5 min. For an isotype control, FLS cells were stained with (Ig)G2a-AF647 (0.1 µg/mL) and 500 nM
SYTOX Green. Positive control staining was performed with HeLa cells. Data were acquired on a FACSAria II Flow Cytometer (BD Biosciences, San Jose, CA) and analysis was
performed using BD FACSDiva 8.0 software. 2.3.8 Statistical Analysis
To analyze single cells in these chemical cytometry experiments, the distribution of the difference between the mean percentage of phosphorylated peptide or intact full length reporter in each single-cell analysis treatment and control group was calculated utilizing bootstrapping.20 10,000 bootstrap replicates (with replacement) were generated from each
group and the means of each bootstrap replicate group were calculated. The distribution of the differences between the appropriate pairs of bootstrap replicates was utilized to estimate the distribution of the mean differences. The p-value for testing the null hypothesis of no difference in mean between the control and treatment groups was estimated by dividing the number of bootstrap differences which possessed values of 0 or less by the number of bootstrap replicates (10,000).
59
Boxplots were utilized to represent the non-normal distribution of the reporter
phosphorylation from individual cells. Within the boxplots, squares represent the mean value from all cells. The middle bar indicates the median, while the upper and lower boxes showed the 75% and 25% percentile of the data, respectively. Whiskers extend to the 5th and 95th percentiles. Any individual data points outside of the whiskers are outliers.