p-Akt and PI3K inhibitor – singe-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 intensities of p-Akt (Ser473), p-Akt (Thr308), 4E-BP, cleaved caspase 3, and ICP27 in HeLa and A549 cells and summaries of apoptotic HeLa cells - A549_pAkt_Ser473_validation.csv (Supplementary Figure 12a) - HeLa_pAkt_Ser473_validation.csv (Supplementary Figure 8a) - HeLa_pAkt_Thr308_two_replicates.csv (Supplementary Figure 8b) - HeLa_PI3KInhibitor_4EBP_secondReplicate.csv (Supplementary Figure 13b) - HeLa_PI3KInhibitor_4EBP.csv (Figure 6c) - HeLa_PI3KInhibitor_cleaved_caspase3_ICP27_secondReplicate.csv (Supplementary Figure 13d) - HeLa_PI3KInhibitor_cleaved_caspase3_ICP27.csv (Figure 6e) - HeLa_PI3KInhibitor_percentage_apoptotic_cells_secondReplicate.csv (Supplementary Figure 13c) - HeLa_PI3KInhibitor_percentage_apoptotic_cells.csv (Figure 6d) HeLa or A549 cells were mock- or HSV-1-infected (indicated by "condition") and either untreated or treated with a PI3K inhibitor (LY294002) or DMSO (indicated by "treatment"). After fixation, the 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 intensities are provided as background-subtracted mean intensity values for the whole-cell. Data cleanup, background subtraction of the intensity values, and classification of cells 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. At 1.5 hpi, cells were treated with 0.1% (v/v) DMSO or 50 µM LY294002 (containing 0.1% (v/v) DMSO). 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.