Facsdiva software 8

Flow cytometry analysis was used to investigate the expression of signaling pathway markers. Briefly, cells were seeded in 24 well plate at 0.5 × 10⁵ cells/ml in serum free media overnight. Cells were treated with nSMase inhibitor GW4869 or DMSO (vehicle) then subjected to stimulation with TNF-α (10 ng/ml) or BSA (vehicle) for 10 min. After stimulation, cells were collected and washed. Cells were then incubated with fixation/permeabilization buffer (cat# 00-5523-00, eBioscience, San Diego, CA, USA) for 20 min in 4 °C, followed by washing and staining with mouse anti-human p-NF-kB P65-PE (20ul/test; cat # 558423; BD Biosciences) and Alexa Fluor 647 mouse anti-IκBα (5ul/test; cat # 560817; BD Biosciences) or control isotypes (mouse IgG2b, κ-PE, Cat# 559529; Mouse IgG1-Alexa Fluor 647, Cat # 560817) for 30 min. The cells were then washed and resuspended in PBS supplemented with 2% FCS for FACS analysis (FACSCanto II; BD Bioscience, San Jose, USA). FACS data analysis was performed using BD FACSDiva Software 8 (BD Biosciences, San Jose, USA). We used unstimulated cells as controls for different treatments.

Cell apoptosis was detected using the Annexin V apoptosis detection kit and propidium iodide (PI) staining for flow cytometry (Sigma-Aldrich, St. Louis, MO, USA). HepG2 cell monolayers were trypsinized; cells were collected by centrifugation (400× g for 5 min), resuspended in 1× binding buffer, and stained using 5 μL Annexin V and 10 μL PI at room temperature for 15 min. Fluorescence-activated cell-sorting (FACS) analysis was carried out using a FACSCanto II flow cytometer (BD Bioscience, San Jose, CA, USA). For each acquisition, 20,000 events were recorded and analyzed. Unstained cells were used to establish the baseline for the negative and positive gates in the analysis, and BD FACSDiva™ Software 8 (BD Biosciences, San Jose, CA, USA) was used for data analysis.

The BrdU Assay was performed according to the manufacturer`s protocol (Roche). 1.5x10⁴ cells/96-well plate were seeded in triplicates in supplemented KBM-Gold medium. After 20 h, cells were serum-starved. 24 h later the medium was replaced by supplemented KBM-Gold with 10 μM BrdU. The incorporation of BrdU was detected after 17 h by anti-BrdU-peroxidase labeled antibody. Enzymatic reaction with TMB (tetramethyl-benzidine) was measured after stopping the reaction with 2 N H₂SO₄ in the Victor II plate reader at 420 nm. For DNA cell cycle analysis, HaCaT pLXSN and HPV8 E6 expressing cells seeded at a cell density of 3x10⁵ in 6-well plates were fixed 4 d later in acetone/methanol for 10 min at -20°C. Cells were treated for 1 h with 1 mg/ml RNase and stained with 5 μg PI per 100 μl cells for 30 min on ice to determine the DNA content by flow cytometry at 488 nm (FACSCanto II, BD Biosciences). The single cell population was gated and cell counts were measured in the PI histogram plot to calculate G0/G1-, S- and G2/M-phases by FACSDiva software 8.0.1 (BD Biosciences). For scratch assay HaCaT monolayers were scratched in 4 cm-dishes. The cells were analyzed at time points 0, 24 and 48 h with Leica DMI600B microscope and Leica application suite software (LAS) V3.06. Cells transfected with p63 siRNA or hsa-miR-203 were scratched 24 h after transfection.

Single-cell suspensions were interrogated using a LSR Fortessa Cell Analyzer flow cytometer (BD Biosciences) with FACS Diva software 8.0.1 (BD Biosciences). Daily baseline performance checks were performed using Cytometry Setup and Tracking Research Beads (BD Biosciences). FlowJo software 10.6.1 (BD Biosciences) was used for analysis. Compensation beads were first used to set correct photomultiplier tubes detectors voltages, then to calculate the amount of fluorescent spill-over to subtract from each channel. Viable cells were selected by separating cells from debris (SSC-A vs FSC-A), gating on single cells (SSC-H vs SSC-W), and finally selecting cells negative for the live/dead viability dye (viability dye vs SSC-A) (Additional File 3).