p-ERK and innate immunity – single-cell feature values
Description
Data in the paper "Cellular state landscape and herpes simplex virus type 1 infection progression are connected" by Pietilä et al., 2023: - Single-cell mean intensity of p-ERK, p-STAT1 (Tyr701), p-STAT1 (Ser727), IRF7, and NF-kB in HeLa cells - Single-cell mean intensity of p-ERK in A549 cells - A549_pERK.csv (Supplementary Figure 11a,b) - HeLa_IRF7.csv (Supplementary Figure 9d) - HeLa_JakInhibitor_pERK.csv (Supplementary Figure 9c) - HeLa_NFkB.csv (Supplementary Figure 9d) - HeLa_pERK_validation.csv (Supplementary Figure 8c) - HeLa_pSTAT1_Ser727.csv (Supplementary Figure 9d) - HeLa_pSTAT1_Tyr701.csv (Supplementary Figure 9d) Cells were mock- or HSV-1-infected and left untreated or treated with IFN-gamma, Jak Inhibitor, or DMSO (indicated by “condition”), and after fixation markers were detected by immunofluorescence imaging. Cells from HSV-1-infected wells were classified into uninfected cells without infected neighbors, uninfected cells with infected neighbors, and infected cells (indicated by “classification_infection”) as described in the paper. Single-cell features are provided as background-subtracted mean intensity values for the nucleus or whole-cell. Data cleanup and background subtraction of the intensity values were applied as described in the paper. Experimental conditions are in duplicate or triplicate (indicated by “well_name”).
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Steps to reproduce
3,500 HeLa cells were seeded per well in 96-well plates and grown at 37°C and 5% CO2 for ~48 h. Cells were then infected with HSV-1 in serum-free DMEM using MOI 0.3. Cells were then incubated with the virus for 30 min at 4°C, and then unbound virus was removed by washing cells with warm DMEM supplemented with 10% (v/v) FBS. Cells were then incubated for 60 min at 37°C, and non-internalized virus was removed by washing cells with acid buffer (40 mM Na citrate, 135 mM NaCl, 10 mM KCl, pH 3.0). Cells were then washed with warm DMEM supplemented with 10% (v/v) FBS and grown at 37°C. At 12 hpi, cells were fixed with 4% paraformaldehyde. After fixation, cells were washed with PBS, and free aldehyde groups were quenched with 500 mM NH4Cl. Permeabilization was done using 0.2% (v/v) Triton X-100 followed by washing with PBS. Cells were then blocked in 3% (w/v) BSA, and after blocking cells were incubated with the primary antibodies, washed with PBS, and then incubated with the secondary antibodies. Antibodies were diluted in 3% (w/v) BSA. After PBS washing, nuclear DNA was stained with NucBlue Fixed Cell ReadyProbes Reagent (DAPI) and total protein was stained with Alexa Fluor 647 NHS Ester (Succinimidyl ester). If cells were stained with anti-pSTAT1 antibodies, a second permeabilization step in 0.1% (w/v) SDS was performed after the Triton X-100 step. Cells were pre-treated with 10 µM Jak Inhibitor (containing 0.1% (v/v) DMSO) or 0.1% (v/v) DMSO 3 h before infection, and then the inhibitor or DMSO was added at 1.5 hpi. Cells were treated with IFN-gamma (100 ng/mL) for 30 min before fixation. Samples were imaged on an automated spinning-disk confocal microscope from Molecular Devices (ImageXpress Confocal HT.ai) using a 20×/NA0.95 water objective, four excitation lasers (405, 470, 555, and 640 nm), and a CMOS camera. Cells and nuclei were segmented and mean intensity of the markers was quantified using maximum intensity projections. Mitotic cells and cells at image borders were removed from the dataset. Computational image analysis was performed using TissueMAPS.