Fig. 5. Co-stimulation of HaCaT cells with IFN-γ and gC increases adhesion of Jurkat cells.

a Schematic representation of the assay. HaCaT cells were seeded 1 day prior to mock-stimulation or stimulation with IFN-γ, gC or both. 24 h after stimulation, Hoechst-labelled Jurkat cells were added and allowed to adhere for 15 min at 37 °C with shaking at 150 rpm. Then, non-adhered cells were washed off and two randomly selected regions of interest (ROI) were imaged per well (triplicate per condition) using an automated microscope (Cytation3, BioTek). The nuclei from adhered cells were quantified using a CellProfiler pipeline. b–d Adhered Jurkat cells per ROI plotted in a bar chart after normalisation to the overall mean of adhered cells from each assay. Depicted are the comparisons between the four treatment conditions (b), the titration of the gC concentration (c), and the comparison of the different gC constructs (d). If not stated otherwise, 5 ng/mL IFN-γ and 300 nM gC were used. Shown is the mean ± SD. Filled circles represent the values from each independent assay (n = 3 biological replicates in b–d). Ordinary one-way ANOVA followed by Šídák’s multiple comparisons test (b, to test for preselected pairs) or followed by Dunnett’s multiple comparisons test (c and d, to test each against a control = IFN-γ) were performed. e Histograms showing expression of lymphocyte function-associated antigen 1 (LFA-1, CD11a) in Jurkat WT (left) and LFA-1 knockout (KO) cells. f Adhesion assay comparing wild-type (WT) Jurkat cells to LFA-1 KO Jurkat cells. Adhered cells per ROI are plotted in a bar chart. Shown is the mean ± SD. Filled circles represent the mean values from each independent assay (n = 3 biological replicates, performed in triplicates with 2 ROI per well). Two-way ANOVA followed by Šídák’s multiple comparisons test (to test between the two cell types) was performed. ns not significant; *P < 0.033; **P < 0.002; ***P < 0.001. Data in b–f were provided as Source Data.